Anti-Resorptive and Bone Building Dietary Supplements and Methods of Use

ABSTRACT

Disclosed herein are dietary supplement compositions and methods for increasing or stimulating bone growth, decreasing or preventing bone resorption, increasing bone strength, improving bone structure, and improving bone architecture comprising a first composition comprising a combination of at least two of: quercetin,  Rehmannia  sp.,  Rehmannia  sp. root, Siberian ginseng,  Sophora japonica , licorice, and ipriflavone, wherein the combination of the first composition increases bone morphogenic protein-2 gene, promoter or protein expression; and a second composition comprising an extract of pomegranate in combination with at least one of Siberian ginseng,  Ginkgo biloba , green tea,  Sophora japonica, Rehmannia  sp., grape seed, Dong Quai, and ipriflavone, wherein the combination of the second composition inhibits the expression of RANK-L.

This application is a divisional of claims priority to U.S. applicationSer. No. 11/977,696 filed Oct. 24, 2007; U.S. Provisional ApplicationSer. No. 60/854,312 filed Oct. 24, 2006; and U.S. ProvisionalApplication Ser. No. 60/925,914 filed Apr. 23, 2007, the entire contentsof which are hereby incorporated by reference.

BACKGROUND

Healthy bones continuously undergo a remodeling process, where anequilibrium is reached between bone resorption and bone formationthrough the concerted action of active bone cells, i.e. bone formingosteoblasts and bone resorbing osteoclasts. The bone remodeling processbegins with activation of cells covering unmineralized bone, i.e. liningcells. The lining cells resorb the unmineralized bone, then retract andleave room for the osteoclasts which resorb the old, mineralized boneand create an environment which attracts the osteoblast to the samesite. The osteoblasts thereafter lay down an organic matrix, whichsubsequently becomes mineralized to form new bone. Thus bone mass isdetermined by the balance between bone resorption by osteoclasts andbone formation by osteoblasts.

The amount of mineral in bone is largely responsible for its hardness,while substances like the structural protein collagen also contribute tobone's mechanical strength. The dense outermost bone is known ascortical bone while the more spongy internal form is known as cancellousor trabecular bone.

Most bone diseases are due to a disruption in the equilibrium of thebone remodeling process. Generally, the disruption is an increase inbone resorption. For example, osteoporosis, one of the most common bonediseases, is characterized by a decrease in bone mass along with amicrostructural change in bone, but there is no effect on the chemicalcomposition of bone itself which results in increased susceptibility tobone fractures. Specifically, the cortical bone becomes thin and porouswhile the trabecular bone becomes thinned, perforated, and disconnected.Osteoporosis may be considered the result of a negative balance in thebone remodeling cycle, i.e. less bone is formed than is being resorbed.

Thus, therapeutic agents for treating bone disorders are directed atinhibiting bone resorption and increasing bone formation. There are manydifferent molecules and pathways involved in the bone remodeling processand the various therapeutic agents presently available target differentmolecules and pathways. For example, bisphosphonates (such as aledronateand risedronate) inhibit bone resorption by blocking osteoclastactivity. Other therapeutic agents seek to inhibit bone resorption byblocking binding to members of the TNF receptor/ligand family, such asReceptor Activator for Nuclear Factor κ B Ligand (RANK-L), a cytokinethat activates osteoclasts, the cells that are involved in boneresorption. Inhibition of release of RANK-L prevents bone mineral loss.

Still other therapeutic agents target increasing bone formation. Forexample, activated bone morphogenic protein gene is known to have directeffects on triggering osteoblast cell differentiation and promoting boneformation. Delivery of recombinant bone morphogenic protein-2 (BMP-2)has been shown to induce bone or cartilage formation. However, systemicadministration of pharmaceutical and biological agents, such asrecombinant BMP-2, can have deleterious effects on the intestine andother tissues. Therefore, there is a need in the art for natural andplant-derived extracts, that can be used in dietary supplementinterventions for preventing and/or treating bone disorders byinhibiting bone resorption and/or increasing bone formation.

BRIEF SUMMARY

The present invention is based on the discovery that novel combinationsof various natural and plant-derived extracts can (1) inhibit boneresorption by inhibiting, decreasing, or preventing the expressionand/or release of RANK-L, and/or by preventing calcium release frombones; (2) increase bone growth by increasing or stimulating gene and/orprotein expression of BMP-2 and (3) improve or maintain bone strength.

In one example, the invention is a composition for increasing orstimulating bone growth comprising natural, plant-derived extracts,including a combination of at least two of the following: quercetindihydrate, quercetin anhydrate, extract of Rehmannia sp., extract ofRehmannia sp. root, extract of Siberian ginseng, extract of Sophorafructus japonica, extract of Sophora japonica, extract of licorice, andipriflavone, wherein the combination increases BMP-2 gene or proteinexpression.

In another example, the invention is a composition for increasing orstimulating bone growth comprising a combination of quercetin anhydrateor dihydrate, an extract of Siberian ginseng, an extract of Sophorajaponica, and an extract of licorice, wherein the combination increasesBMP-2 gene or protein expression.

In another example, the invention is a composition for increasing orstimulating bone growth comprising a combination of approximately10-1000 mg of quercetin anhydrate or dihydrate, approximately 100-800 mgof an extract of Siberian ginseng, and approximately 10-500 mg of anextract of licorice, wherein the combination increases BMP-2 gene orprotein expression.

In a further example, the invention is a composition for increasing orstimulating bone growth comprising a combination of approximately10-1000 mg of quercetin anhydrate or dihydrate, and approximately 10-500mg of an extract of licorice, wherein the combination increases BMP-2gene or protein expression.

In another example, the invention is a composition for increasing orstimulating bone growth comprising a combination of approximately10-1000 mg of quercetin anhydrate or dihydrate, and approximately100-800 mg of an extract of Siberian ginseng, wherein the combinationincreases BMP-2 gene or protein expression.

In a further example, the invention is a method of increasing orstimulating bone growth in a subject, comprising administering to thesubject a composition comprising a combination of at least two of thefollowing: quercetin anhydrate, quercetin dihydrate, extract ofRehmannia sp., extract of Rehmannia sp. root, extract of Siberianginseng, extract of Sophora japonica, extract of licorice, andipriflavone, wherein the combination increases BMP-2 gene or proteinexpression in the subject.

A further example of the invention is a method of increasing orstimulating bone growth in a subject, comprising administering to thesubject a composition comprising a combination of approximately 10-1000mg of quercetin anhydrate or dihydrate, approximately 100-800 mg of anextract of Siberian ginseng, and approximately 10-500 mg of an extractof licorice, wherein the combination increases BMP-2 gene or proteinexpression in the subject.

Another example of the invention is a method of increasing orstimulating bone growth in a subject, comprising administering to thesubject a composition comprising a combination of approximately 10-1000mg quercetin anhydrate or dihydrate, and approximately 10-500 mg of anextract of licorice, wherein the combination increases BMP-2 gene orprotein expression in the subject.

A further example of the invention is a method of increasing orstimulating bone growth in a subject, comprising administering to thesubject a composition comprising a combination of approximately 10-1000mg quercetin anhydrate or dihydrate, and approximately 100-800 mg of anextract of Siberian ginseng, wherein the combination increases BMP-2gene or protein in the subject.

In another example, the present invention may be a composition forinhibiting, decreasing, or preventing bone resorption comprising apomegranate extract, punicalagins, or both, in combination with one ormore of the following: quercetin dihydrate, quercetin anhydrate, extractof Rehmannia sp., extract of Rehmannia sp. root, extract of Siberianginseng, extract of Sophora fructus japonica, extract of Sophorajaponica, extract of licorice, and ipriflavone, wherein the pomegranateextract, the punicalagins, or both inhibit expression, production,and/or release of RANK-L.

In one example, compositions and methods of the present inventionutilize extracts of pomegranate (Punica granatum) fruit and peel whichcontain compounds known as punicalagins, to inhibit or decrease boneresorption. The extracts of pomegranate useful in the present invention,for example a pomegranate extract comprised of punicalagins, can be usedin compositions and methods for inhibiting, decreasing, or preventingbone resorption, wherein the pomegranate extract inhibits, decrease orprevent the expression, production, and/or release of RANK-L.Accordingly, in one example the present invention is a composition forinhibiting, decreasing, or preventing bone resorption that comprises apomegranate extract, at least one punicalagin, or both. Alternatively,the present invention contemplates a method of inhibiting, decreasing,or preventing bone resorption comprising administering a compositioncomprising a pomegranate extract, at least one punicalagin, or both,wherein the composition inhibits one of expression, production, and/orrelease of RANK-L.

In yet another example, the present invention is a composition forinhibiting, decreasing, or preventing bone resorption comprisingnatural, plant-derived extracts, including a combination of at least twoof the following: an extract of pomegranate preferably containingpunicalagins, an olive extract, an extract of Siberian ginseng, anextract of Ginkgo biloba, an extract of green tea, an extract of Sophorajaponica, an extract of Rehmannia sp., an extract of grape seed, anextract of Dong Quai, and ipriflavone, wherein the combination inhibitsexpression, production, and/or release of RANK-L.

In a further example, the present invention is a composition forinhibiting, decreasing, or preventing bone resorption comprising acombination of an extract of pomegranate, an extract of grape seed,ipriflavone, and an extract of green tea, wherein the combinationinhibits expression, production, and/or release of RANK-L.

In another example, the present invention is a composition forinhibiting, decreasing, or preventing bone resorption comprising acombination of approximately 10-2000 mg of an extract of pomegranate,approximately 35-250 mg of an extract of grape seed, and approximately400-700 mg of ipriflavone, wherein the combination inhibits expression,production, and/or release of RANK-L.

In yet another example, the present invention is a composition forinhibiting, decreasing, or preventing bone resorption comprisingapproximately 10-2000 mg of an extract of pomegranate, approximately35-250 mg of an extract grape seed, and approximately 400-700 mg ofipriflavone, wherein the composition inhibits release of calcium frombones.

Another example of the invention is a method of inhibiting, decreasing,or preventing bone resorption in a subject comprising administering tothe subject a composition comprising natural, plant-derived extracts,including a combination of at least two of the following extracts: anextract of pomegranate, an olive extract, an extract of Siberianginseng, an extract of Ginkgo biloba, an extract of green tea, anextract of Sophora japonica, an extract of Rehmannia sp., an extract ofgrape seed, an extract of Dong Quai, and ipriflavone, wherein thecombination inhibits expression, production, and/or release of RANK-L.

In a further example, the invention is a method of inhibiting,decreasing, or preventing bone resorption in a subject comprisingadministering to the subject a composition comprising a combination ofan extract of pomegranate, an extract of grape seed, ipriflavone, and anextract of green tea, wherein the combination inhibits expression,production, and/or release of RANK-L.

In a further example, the invention is a method of inhibiting,decreasing, or preventing bone resorption in a subject comprisingadministering to the subject a composition comprising a combination ofapproximately 10-2000 mg of an extract of pomegranate, approximately35-250 mg of an extract of grape seed, and approximately 400-700 mg ofipriflavone, wherein the combination inhibits expression, production,and/or release of RANK-L.

In yet another example, the invention is a method of inhibiting,decreasing, or preventing bone resorption in a subject comprisingadministering to the subject a composition comprising a combination ofapproximately 10-2000 mg of an extract of pomegranate, approximately35-250 mg of an extract of grape seed, and approximately 400-700 mg ofipriflavone, wherein the combination inhibits release of calcium frombones.

In a further example, the invention is a dietary supplement regimen forincreasing or stimulating bone growth and inhibiting, decreasing, orpreventing bone resorption comprising a first composition comprising acombination of at least two of quercetin dihydrate, quercetin anhydrate,an extract of Rehmannia sp., an extract of Rehmannia sp. root, anextract of Siberian ginseng, an extract of Sophora japonica, an extractof licorice, and ipriflavone, wherein the combination of the firstcomposition increases the expression and/or activity of BMP-2; and asecond composition comprising a combination of at least two of anextract of pomegranate, an olive extract, an extract of Siberianginseng, an extract of Ginkgo biloba, an extract of green tea, anextract of Sophora japonica, an extract of Rehmannia sp., an extract ofgrape seed, an extract of Dong Quai, and ipriflavone, wherein thecombination of the second composition inhibits the expression of RANK-L.

DETAILED DESCRIPTION

It is to be understood that this invention is not limited to theparticular compositions, methodology, or protocols described herein.Further, unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood to one ofordinary skill in the art to which this invention belongs. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which will be limited only by theclaims.

The present invention is based on the surprising discovery that uniquecombinations of ingredients including two or more of the following:quercetin anhydrate, quercetin dihydrate, extract of Rehmannia sp.,extract of Rehmannia sp. root, extract of Siberian ginseng, extract ofSophora japonica, extract of licorice, extract of ipriflavone, extractof pomegranate, extract of olive, extract of Ginkgo biloba, extract ofgreen tea, extract of grape seed, and extract of Dong Quai, which aredescribed more fully below, increase bone growth by increasing orstimulating expression and/or activity of the BMP-2 promoter, gene, andor/protein, or inhibit bone resorption by either inhibiting, decreasing,or preventing RANK-L expression, production, or release or byinhibiting, decreasing, or preventing release of calcium from bones.

BMP-2 is a member of a family of bone morphogenic proteins, which arenovel factors in the extended transforming growth factor B superfamily.Recombinant BMP-2 and BMP-4 can induce new bone formation when injectedlocally into the subcutaneous tissues of rats (Wozney J. Molec (1992)32: 160-67). BMP-2 and BMP-4 are expressed by normal osteoblasts as theydifferentiate, and have been shown to stimulate osteoblastdifferentiation and bone nodule formation in vitro as well as boneformation in vivo. Thus, by increasing or stimulating BMP-2 promoteractivity, gene expression, and/or protein expression, the uniquecompositions of the present invention are useful for increasing orstimulating bone growth and treating or preventing a variety of bonedisorders.

RANK-L, receptor activator of nuclear factor (NF)-kB ligand (also:Osteoprotegerin ligand, OPGL) a member of the Tumor Necrosis Factor(TNF) family, is the main stimulatory factor for the formation of matureosteoclasts, bone cells that aid in bone resorption, and is essentialfor their survival. RANK-L is produced by osteoblastic lineage cells andactivated T lymphocytes. It activates the specific receptor RANK that islocated on osteoclasts and dendritic cells. One strategy for aformulation of the present invention is to directly inhibit RANK-Lexpression when stimulated by IL-1. Activation of RANK-L when stimulatedwith IL-1 can directly trigger osteoclastogenesis (and thus boneresorption). Thus, by inhibiting, decreasing, or preventing RANK-Lexpression, production, or release, the unique compositions of thepresent invention are useful for inhibiting, decreasing, or preventingbone resorption and treating or preventing a variety of bone disordersincluding bone loss, osteoporosis, osteolytic bones, etc.

Quercetin, which refers to quercetin extract(s) and the various forms ofquercetin, such as quercetin dihydrate, quercetin anhydrate, etc., isone compound that is useful in unique compositions of the presentinvention. Quercetin is a flavonoid that forms the “backbone” for manyother flavonoids, including the citrus flavonoids rutin, hesperidin,naringin and tangeritin. Quercetin is found to be the most active of theflavonoids in studies, and many medicinal plants owe much of theiractivity to their high quercetin content. Quercetin has demonstratedsignificant anti-inflammatory activity because of direct inhibition ofseveral initial processes of inflammation. For example, it inhibits boththe manufacture and release of histamine and other allergic/inflammatorymediators. In addition, it exerts potent antioxidant activity andvitamin C-sparing action.

Quercetin also may have positive effects in combating or helping toprevent cancer, prostatitis, heart disease, cataracts, allergies,inflammations, and respiratory diseases such as bronchitis and asthma.In addition, according to U.S. Pat. No. 5,478,579, when used in amountsranging from 50-1500 mg/day, quercetin anhydrate and/or quercetindihydrate can enhance absorption of calcium into bone tissues.

Foods rich in quercetin include apples, black & green tea, onions,raspberries, red wine, red grapes, citrus fruits, broccoli, fava beans,other leafy green vegetables, and cherries.

As discussed more fully below, the present invention is based in part onthe discovery that quercetin is a potent activator of BMP-2 promoteractivity and protein expression. In addition, the assay resultsdiscussed below demonstrate that when quercetin dihydrate isadministered in combination with Siberian ginseng, licorice, or both,the combination achieves a surprising synergy resulting in BMP-2promoter activity and protein expression beyond that achieved with anyingredient alone.

A quercetin ingredient used in the present invention may be obtainedcommercially from various sources including, for example, Twinlab(American Fork, Utah), Jarrow Formulas (Los Angeles, Calif.), NaturalFactors (Coquitlam, British Columbia, Canada), and NOW Foods(Bloomingdale, Ill.). In addition, quercetin may be obtained by any ofthe extraction methods discussed more fully below, or described or knownin the art.

Rehmannia, another plant useful in unique compositions of the presentinvention, is a genus of six species of flowering plants in the orderLamiales, endemic to China. Known as dihuang (

) in Chinese, this medicinal herb is used for a variety of ailments suchas anemia, dizziness and constipation. Rehmannia contains the vitaminsA, B, C, and D, as well as other useful compounds.

Rehmannia sp. extracts or extracts of Rehmannia sp., including extractsof Rehmannia sp. roots or Rehmannia sp. root extracts may be obtainedcommercially from various sources including EUL Herb Manufacturing (LaVerne, Calif.) and NuPharma Neutraceuticals (Miami Beach, Fla.). Inaddition, extracts of Rehmannia sp. may be obtained by any of theextraction methods discussed more fully below or known or described inthe art.

Siberian ginseng, one of the compounds useful in unique compositions ofthe present invention, which is also known as Eleutherococcussenticosus, is a species of small, woody shrub in the family Araliaceaenative to Northeastern Asia. Siberian ginseng is a powerful tonic herbwith a wide range of health benefits. For example, Siberian ginseng hasimmunoprotective effects against breast (mammary gland) carcinoma,stomach carcinoma, oral cavity carcinoma, skin melanoma and ovariancarcinoma. It was found to have a pronounced effect on T lymphocytes,predominantly of the helper/inducer type, but also on cytotoxic andnatural killer cells. In addition, Siberian ginseng is known to have aprotective effect against osteoporosis. See, e.g., Kropotov et al.,“Effects of Siberian ginseng extract and ipriflavone on the developmentof glucocorticoid-induced osteoporosis.” Bull Exp Biol Med., 2002.133(3):252-4.

As discussed more fully below, the present invention is based in part onthe discovery that extracts of Siberian ginseng (or Siberian ginsengextracts) are potent activators of BMP-2 promoter activity, BMP-2 geneexpression and BMP-2 protein expression. In addition, the assay resultsdiscussed below demonstrate that when extracts of Siberian ginseng areadministered in combination with quercetin dihydrate, the combinationachieves a surprising synergy resulting in BMP-2 promoter activity, geneexpression and protein expression beyond that achieved with eitheringredient alone.

Siberian ginseng extracts may be commercially obtained from varioussuppliers such as Xi'an Tianxingjian Natural Bio-products Group (Xi'an,Shaanxi, China). In addition, Siberian ginseng extract may be obtainedusing any of the extraction techniques discussed more fully below orknown in the art. In one example, a Siberian ginseng extract may beobtained as an alcoholic fluid extraction of the root and/or rhizome ofSiberian ginseng.

Sophora japonica, or Sophora fructus japonica, another of the compoundsuseful in unique compositions of the present invention, is also referredto as the Pagoda Tree and is native to eastern Asia. Rutin, an activecompound that may be found in a Sophora japonica extract, may be used toincrease the permeability (e.g. the resolution and porousness of thedilation) of capillaries. In addition to rutin, quercetin anhydrate andquercetin dihydrate may be extracted from Sophora japonica plants,including from the leaves, stem, flower, seeds, root, etc.

As discussed more fully below, the present invention is based in part onthe discovery that extracts of Sophora japonica (or Sophora japonicaextracts) are potent activators of BMP-2 promoter activity, geneexpression and protein expression.

Sophora japonica extracts may be commercially obtained from varioussuppliers such as NuPharma Nutraceuticals (Miami Beach, Fla.). Inaddition, Sophora japonica extract may be obtained using any of theextraction techniques discussed more fully below or known in the art. Inone example, the ripe seeds of Sophora japonica may be used to obtain aSophora japonica extract useful in the present invention.

Licorice extracts, useful in unique compositions of the presentinvention, are widely used in treating bronchial problems such ascatarrh, bronchitis and coughs in general. Licorice also forms animportant ingredient in controlling peptic ulcerations, gastritis andulcers. According to JP 2002 179585 and US 20020009506, whenadministered in an amount ranging from 50-100 mg per day, licorice canbe used to treat osteoporosis, improve bone metabolism, and promotecalcification.

As discussed more fully below, the present invention is based in part onthe discovery that licorice extracts (or extracts of licorice), forexample ethanol and ethanol-water extracts of licorice, are potentactivators of BMP-2 promoter activity, gene expression and proteinexpression. In addition, the assay results discussed below demonstratethat when licorice extract is administered in combination with quercetindihydrate, the combination achieves a surprising synergy resulting inBMP-2 promoter activity, gene expression and protein expression beyondthat achieved with either ingredient alone.

Licorice extracts may be commercially obtained from various supplierssuch as Herbs Forever, Inc. (Los Angeles, Calif.). In addition, alicorice extract may be obtained using any of the extraction techniquesdiscussed more fully below or known in the art. In one example, alicorice extract can be an ethanol extract of licorice obtained from theroot, runner, and/or rhizome of Glycyrrhiza glabra.

Ipriflavone, another of the compounds useful in unique compositions ofthe present invention, is an isoflavone. Although Ipriflavone may befound in small amounts in legume plants, such as alfalfa, Ipriflavonegenerally is synthetically manufactured as 7-isopropoxy isoflavone,using polyphenols as a starting material.

As discussed more fully below, the present invention is based in part onthe discovery that ipriflavone is a potent inhibitor of RANK-Lexpression. Ipriflavone may be commercially obtained from varioussources. For example, Ostivone® is a synthetic ipriflavone compoundavailable from Technical Sourcing International, Inc. (Missoula, Mont.).

Pomegranates can be extracted to yield an extract of pomegranate(pomegranate extract) that is useful in unique compositions of thepresent invention. When extracted, pomegranate, known as Punicagranatum, is generally standardized to ellagic acid or punicalagincontent. Punicalagins exist as isomers of2,3,hexahydroxydiphenoyl-gallagyl-D-glucose. An exemplary structure isshown below:

Extracts of pomegranate also may be high in polyphenols, such ashydrolysable tannins, and particularly punicalagins, which may beresponsible for the free-radical scavenging ability of pomegranatejuice.

In Japan, pomegranate has been used to inhibit bone quantity reduction.See JP 1999 0049884. The present invention is based in part on thediscovery that an extract of pomegranate is a potent inhibitor of RANK-Lexpression, production, or release. In one example, punicalagins presentin extracts of pomegranate inhibit or decrease RANK-L expression.Therefore, pomegrantate extracts comprising punicalagins, ellagic acidor both are useful in compositions and methods for inhibiting,decreasing, or preventing the production, release, and/or expression ofRANK-L.

Pomegranate extracts may be commercially obtained from various sourcesincluding Nature's Way (Springville, Utah), Nature's Herbs (AmericanFork, Utah), Swansen's Health Products (Fargo, N. Dak.) and Doctor'sTrust Vitamins (Orlando, Fla.). In addition, a pomegranate extract maybe obtained using any of the extraction techniques discussed more fullybelow or known in the art.

Extracts of Ginkgo biloba (or Ginkgo biloba extracts), another of theextracts useful in unique compositions of the present invention, areknown to have three effects: (1) improve blood flow (includingmicrocirculation in small capillaries) to most tissues and organs; (2)protect against oxidative cell damage from free radicals (antioxidant);and (3) block the effects of platelet aggregation and blood clotting.

Ginkgo biloba extracts may be commercially obtained from various sourcesincluding Puritan's Pride (Long Island, N.Y.). In addition, Ginkgobiloba extracts may be obtained using any of the extraction processdisclosed herein or known in the art. For example, Ginkgo bilobaextracts may be obtained using any of the extraction processes disclosedherein or known in the art to extract dried or fresh leaves, or seeds ofGingko biloba.

Green Tea, which can be extracted to yield one of the compounds usefulin unique compositions of the present invention, has long been used bythe Chinese as medicine to treat headaches, body aches, poor digestion,and improve well-being and life expectancy. Green tea extract is rich inbioflavonoids, including the anti-oxidant epigallocatechin gallate(EGCG). The EGCG in green tea extract protects against digestive andrespiratory infections, blocks the actions of carcinogens, can functionas an anti-bacterial, and can also help lower cholesterol levels.

As discussed more fully below, the present invention is based in part onthe discovery that green tea extract (or an extract of green tea) is apotent inhibitor of RANK-L expression. Green tea extracts may becommercially obtained from various sources including Life Extension(Fort Lauderdale, Fla.). In addition, a green tea extract may beobtained using any of the extraction techniques discussed more fullybelow or known in the art.

Grape Seed extracts (or extracts of grape seeds), another of thecompounds useful in unique compositions of the present invention,contain a class of flavonoid complexes known as oligomericproanthocyanidins or OPCs that act as antioxidants (free radicalscavengers) in the human body. OPCs may help protect against the effectsof internal and environmental stresses (that is, cigarette smoking,pollution, and supporting normal body metabolic processes).

The extracts of grape seed used in the present invention may be obtainedfrom commercially available sources. For example, the grape seed extractmay be obtained from Kikkoman Corporation (Tokyo, Japan), Polyphenolics,Inc. (Madera, Calif.), Bio Serae Laboratories SA (Bram, France),OptiPure (Los Angeles, Calif.), Dry Creek Nutrition, Inc. (Modesto,Calif.), or other suitable sources. In addition, the extractiontechniques discussed more fully below, or those known or described inthe art may be used to produce a grape seed extract to be used in thepresent invention.

Dong Quai, one of the extract useful in unique compositions of thepresent invention, also is known as Angelica sinensis or “femaleginseng” and is an herb from the family Apiaceae, indigenous to China.Its root is commonly known in Chinese as dong quai or danggui (Chinese:

; pinyin: dāngguī) and is widely used in Chinese traditional medicine totreat gynecological ailments, fatigue, mild anemia and high bloodpressure. Dong Quai has analgesic, anti-inflammatory, antispasmodic andsedative effects. The plant's phytochemicals consist of coumarins,phytosterols, polysaccharides, ferulate, and flavonoids.

Dong Quai extract (or extract of Dong Quai) may be commercially obtainedfrom a variety of different sources, including Capricorns Lair (Ogden,Utah). In addition, Dong Quai can be extracted using any of theextraction techniques described more fully below, or any extractiontechniques known in the art.

Although each of the extracts used in the present invention iscommercially available, there are numerous extraction methods that canbe used to produce an extract to be used in the present inventionwithout commercially purchasing the extract. Some examples of extractionmethods that can be used to produce an extract to be used in the presentinvention are described below. Other examples are known and described inthe art, including in various publications and patents. The extractionmethods described more fully below are exemplary and one of ordinaryskill in the art will appreciate that other extraction techniques andmethods may be used to obtain an extract useful in the presentinvention.

Extracts used in the present invention may be from a variety of sources,including different varieties and species. For example, grape seeds fromgrapes of any color or variety may be used to obtain a grape seedextract. In addition, any of the parts of a plant may be extracted,including the fruit, peel, seeds, stem, leaves, roots, bark, rhizome,runner, etc.

In one example, an extract useful in the unique compositions of thepresent invention might be obtained using an organic solvent extractiontechnique. More specifically, an extract useful in the presentinvention, such as a licorice extract or a licorice root extract, can beproduced by extracting licorice or licorice root with an organicsolvent, for example, hexane, ethyl acetate, ethanol, or hydro-ethanol.

In another example, solvent sequential fractionation may be used toobtain an extract useful in the unique compositions of the presentinvention. For example, using this technique, a grape seed extract couldbe obtained by sequentially extracting grape seeds with hexane, ethylacetate, ethanol, and hydro-ethanol. The extracts obtained after eachstep (fractions) of the sequence will contain chemical compounds inincreasing order of polarity similar to the solvents used for extractingthem. The fractions are dried to evaporate the solvents, resulting in anextract of grape seed. Those of skill in the art will appreciate thatmany other solvents can be used in practicing solvent sequentialfractionation extraction.

Total hydro-ethanolic extraction techniques might also be used to obtainan extract useful in the unique compositions of the present invention.Generally, this is referred to as a lump-sum extraction. The extractgenerated in this process will contain a broad variety of phytochemicalspresent in the extracted material including fat and water solubles.Following collection of the extract solution, the solvent will beevaporated, resulting in the extract. In one example, pomegranates mightbe extracted using this technique.

Total ethanol extraction may also be used in the present invention. Thistechnique uses ethanol, rather than hydro-ethanol, as the solvent. Thisextraction technique generates an extract that may include fat solubleand/or lipophilic compounds in addition to water soluble compounds. Anextract of green tea might be obtained using this technique.

Another example of an extraction technique that might be used to obtainan extract useful in the present invention is supercritical fluid carbondioxide extraction (SFE). In this extraction procedure the material tobe extracted is not exposed to any organic solvents. Rather, theextraction solvent is carbon dioxide, with or without a modifier, insupercritical conditions (>31.3° C. and >73.8 bar). Those of skill inthe art will appreciate that temperature and pressure conditions can bevaried to obtain the best yield of extract. This technique generates anextract of fat soluble and/or lipophilic compounds, similar to the totalhexane and ethyl acetate extraction technique described above.

Those of skill in the art will appreciate that there are many otherextraction processes, both known in the art and described in variouspatents and publications that can be used to obtain the extracts to beused in practicing the present invention. For example, the extractionprocedures described in the following references, which are incorporatedherein by reference, could be used in practicing the present invention:Murga et al., “Extraction of natural complex phenols and tannins fromgrape seeds by using supercritical mixtures of carbon dioxide andalcohol.” J. Agric Food Chem. 2000 August: 48(8):3408-12; Hong et al.,“Microwave-assisted extraction of phenolic compounds from grape seed.”Nat Prod Lett. 2001; 15(3):197-204; Ashraf-Khorassani et al.,“Sequential fractionation of grape seeds into oils, polyphenols, andprocyanidins via a single system employing CO₂-based fluids.” J. AgricFood Chem., 2004 May 5; 52(9):2440-4.

COMPOSITIONS OF THE INVENTION

Compositions of the present invention may be formulated in an acceptablecarrier and may be prepared, packaged, and labeled for increasing orstimulating bone growth, inhibiting, decreasing, or preventing boneresorption, increasing bone strength, improving bone structure,improving bone architecture, or treatment, prevention, or management ofvarious bone disorders including, but not limited to, fractures,osteoporosis, periodontal disease, metastatic bone disease, andosteolytic bone disease.

In one example, the invention is a composition for increasing orstimulating bone growth, comprising a combination of at least two of thefollowing: quercetin anhydrate, quercetin dihydrate, Rehmannia sp.extract, Siberian ginseng extract, Sophora japonica extract, licoriceextract, and ipriflavone, wherein the combination increases BMP-2 geneor protein expression.

In another example, the invention is a composition for increasing bonegrowth, comprising a combination of at least two of the following:quercetin, anhydrate, quercetin dihydrate, Rehmannia sp. extract,Siberian ginseng extract, Sophora japonica extract, licorice extract,and ipriflavone, wherein the combination increases BMP-2 gene or proteinexpression, and further wherein, if present: quercetin anhydrate orquercetin dihydrate, is present in an amount ranging from approximately10-1000 mg, more preferably in an amount ranging from approximately200-750 mg, more preferably in an amount ranging from approximately300-700 mg, more preferably in an amount ranging from approximately400-600 mg, more preferably in an amount of approximately 500 mg;Rehmannia sp. extract is present in an amount ranging from approximately10-1000 mg, more preferably in an amount ranging from approximately100-900 mg, more preferably in an amount ranging from approximately200-800 mg, more preferably in an amount ranging from approximately300-700 mg, more preferably in an amount ranging from approximately400-600 mg, more preferably in an amount of approximately 500 mg;Siberian ginseng extract is present in an amount ranging from 100-800mg, more preferably in an amount ranging from approximately 200-750 mg,more preferably in an amount ranging from approximately 300-700 mg, morepreferably in an amount ranging from approximately 400-600 mg, morepreferably in an amount of approximately 500 mg; Sophora japonicaextract is present in an amount ranging from 10-1000 mg, more preferablyin an amount ranging from approximately 100-900 mg, more preferably inan amount ranging from approximately 200-800 mg, more preferably in anamount ranging from approximately 300-700 mg, more preferably in anamount ranging from approximately 400-600 mg, more preferably in anamount of approximately 500 mg; licorice extract is present in an amountranging from approximately 10-500 mg, more preferably in an amountranging from approximately 25-450 mg, more preferably in an amountranging from approximately 50-400 mg, more preferably in an amountranging from approximately 75-350 mg, more preferably in an amountranging from approximately more preferably in an amount ranging fromapproximately 100-300 mg, more preferably in an amount ranging fromapproximately 125-250 mg, more preferably in an amount ranging fromapproximately 25-175 mg; and ipriflavone is present in an amount rangingfrom 200-700 mg, more preferably in an amount ranging from approximately250-650 mg, more preferably in an amount ranging from approximately300-600 mg, more preferably in an amount ranging from approximately400-500 mg, more preferably in an amount of approximately 600 mg.

In another example, the invention is a composition for increasing orstimulating bone growth comprising a combination of quercetin anhydrate,quercetin dihydrate, Siberian ginseng extract, Sophora japonica extract,and licorice extract, wherein the combination increases BMP-2 gene orprotein expression, and further wherein the quercetin anhydrate orquercetin dihydrate is present in an amount ranging from approximately10-1000 mg, more preferably in an amount ranging from approximately200-750 mg, more preferably in an amount ranging from approximately300-700 mg, more preferably in an amount ranging from approximately400-600 mg, more preferably in an amount of approximately 500 mg;Siberian ginseng extract is present in an amount ranging fromapproximately 100-800 mg, more preferably in an amount ranging fromapproximately 200-750 mg, more preferably in an amount ranging fromapproximately 300-700 mg, more preferably in an amount ranging fromapproximately 400-600 mg, more preferably in an amount of approximately500 mg; Sophora japonica extract is present in an amount ranging fromapproximately 10-1000 mg, more preferably in an amount ranging fromapproximately 100-900 mg, more preferably in an amount ranging fromapproximately 200-800 mg, more preferably in an amount ranging fromapproximately 300-700 mg, more preferably in an amount ranging fromapproximately 400-600 mg, more preferably in an amount of approximately500 mg; and licorice extract is present in an amount ranging from 10-500mg, more preferably in an amount ranging from approximately 25-450 mg,more preferably in an amount ranging from approximately 50-400 mg, morepreferably in an amount ranging from approximately 75-350 mg, morepreferably in an amount ranging from approximately 100-300 mg, morepreferably in an amount ranging from approximately 125-250 mg, morepreferably in an amount ranging from approximately 25-175 mg.

In another example, the invention is a composition for increasing orstimulating bone growth comprising a combination of approximately10-1000 mg of quercetin anhydrate, quercetin dihydrate, more preferablyapproximately 250-750 mg, more preferably approximately 300-700 mg, morepreferably approximately 400-600 mg, more preferably approximately 500mg; approximately 100-800 mg of Siberian ginseng, more preferablyapproximately 200-750 mg, more preferably approximately 300-700 mg, morepreferably approximately 400-600 mg, more preferably approximately 500mg; and approximately 10-500 mg of licorice extract, more preferablyapproximately 25-450 mg, more preferably approximately 50-400 mg, morepreferably approximately 75-350 mg, more preferably approximately100-300 mg, more preferably approximately 125-250 mg, more preferablyapproximately 25-175 mg, wherein the combination increases BMP-2 gene orprotein expression.

In a further example, the invention is a composition for increasing orstimulating bone growth comprising a combination of quercetin anhydrateor quercetin dihydrate, Siberian ginseng extract, and licorice extract,wherein the combination increases BMP-2 gene or protein expression, andfurther wherein the quercetin anhydrate or quercetin dihydrate, Siberianginseng extract, and licorice extract are present in equal amounts, morepreferably wherein the Siberian ginseng extract is present in an amountthat is ½ that of quercetin anhydrate or quercetin dihydrate and thelicorice extract is present in an amount that is 1/10 that of quercetinanhydrate or quercetin dihydrate.

In another example, the invention is a composition for increasing orstimulating bone growth comprising a combination of quercetin anhydrateor quercetin dihydrate, and Siberian ginseng extract, wherein quercetinanhydrate or quercetin dihydrate forms approximately 10-75% w/w of thecomposition, more preferably approximately 50% w/w of the compositionand Siberian ginseng extract forms approximately 10-75% w/w of thecomposition, more preferably approximately 50% w/w of the composition,wherein the combination of quercetin anhydrate or quercetin dihydrateand Siberian ginseng extract increases BMP-2 gene or protein expression.

In yet another example, the invention is a composition for increasing orstimulating bone growth comprising a combination of at least two of:quercetin anhydrate, quercetin dihydrate, Siberian ginseng extract,Sophora japonica extract, and licorice extract, wherein if present,quercetin anhydrate or quercetin dihydrate forms approximately 10-75%w/w of the composition, more preferably approximately 50% w/w of thecomposition; Siberian ginseng extract forms approximately 10-75% w/w ofthe composition, more preferably approximately 50% w/w of thecomposition; Sophora japonica extract forms approximately 1-50% w/w ofthe composition, more preferably approximately 2-15% w/w of thecomposition, more preferably approximately 5-10% w/w of the composition;and licorice extract forms approximately 1-50% w/w of the composition,more preferably approximately 2-15% w/w of the composition, morepreferably approximately 5-10% w/w of the composition, further whereinthe combination increases BMP-2 gene or protein expression.

In a further example, the invention is a composition for increasing orstimulating bone growth comprising a combination of approximately10-1000 mg of quercetin anhydrate or quercetin dihydrate, morepreferably approximately 250-750 mg, more preferably approximately300-700 mg, more preferably approximately 400-600 mg, more preferablyapproximately 500 mg; and approximately 10-500 mg of licorice extract,more preferably approximately 20-125 mg, more preferably approximately20-100 mg, more preferably approximately 25-75 mg, more preferablyapproximately 50 mg, wherein the combination increases BMP-2 gene orprotein expression.

In another example, the invention is a composition for increasing orstimulating bone growth comprising a combination of approximately10-1000 mg of quercetin anhydrate or quercetin dihydrate, morepreferably approximately 250-750 mg, more preferably approximately300-700 mg, more preferably approximately 400-600 mg, more preferablyapproximately 500 mg; and approximately 100-800 mg of Siberian ginsengextract, more preferably approximately 200-750 mg, more preferablyapproximately 300-700 mg, more preferably approximately 400-600 mg, morepreferably approximately 500 mg, wherein the combination of quercetinanhydrate or quercetin dihydrate, and Siberian ginseng extract increasesBMP-2 gene or protein expression.

In a further example, the invention is a method of increasing orstimulating bone growth in a subject, comprising administering to thesubject a composition comprising a combination of at least two of thefollowing: quercetin anhydrate, quercetin dihydrate, Rehmannia sp.extract, Rehmannia sp. root extract, Siberian ginseng extract, Sophorajaponica extract, licorice extract, and ipriflavone, wherein thecombination increases BMP-2 gene or protein expression in the subject.

A further example of the invention is a method of increasing orstimulating bone growth in a subject, comprising administering to thesubject a composition comprising a combination of approximately 10-1000mg of quercetin anhydrate or quercetin dihydrate, more preferablyapproximately 250-750 mg, more preferably approximately 300-700 mg, morepreferably approximately 400-600 mg, more preferably approximately 500mg; approximately 100-800 mg of Siberian ginseng extract, morepreferably approximately 200-750 mg, more preferably approximately300-700 mg, more preferably approximately 400-600 mg, more preferablyapproximately 500 mg; and approximately 10-500 mg of licorice extract,more preferably approximately 25-450 mg, more preferably approximately50-400 mg, more preferably approximately 75-350 mg, more preferablyapproximately 100-300 mg, more preferably approximately 125-250 mg, morepreferably approximately 25-75 mg, wherein the combination increasesBMP-2 gene or protein expression.

Another example of the invention is a method of increasing orstimulating bone growth in a subject, comprising administering to thesubject a composition comprising a combination of approximately 10-1000mg quercetin anhydrate or quercetin dihydrate, more preferablyapproximately 250-750 mg, more preferably approximately 300-700 mg, morepreferably approximately 400-600 mg, more preferably approximately 500mg; and approximately 10-500 mg of licorice extract, more preferablyapproximately 20-125 mg, more preferably approximately 20-100 mg, morepreferably approximately 25-75 mg, more preferably approximately 50 mg,wherein the combination increases BMP-2 gene or protein expression.

A further example of the invention is a method of increasing orstimulating bone growth in a subject, comprising administering to thesubject a composition comprising a combination of approximately 10-1000mg quercetin anhydrate or quercetin dihydrate, more preferablyapproximately 250-750 mg, more preferably approximately 300-700 mg, morepreferably approximately 400-600 mg, more preferably approximately 500mg; and approximately 100-800 mg of Siberian ginseng extract, morepreferably approximately 200-750 mg, more preferably approximately300-700 mg, more preferably approximately 400-600 mg, more preferablyapproximately 500 mg, wherein the combination of quercetin anhydrate orquercetin dihydrate, and Siberian ginseng extract increases theexpression and/or activity of the BMP-2 gene or protein in the subject.

In another example, the present invention is a composition forinhibiting, decreasing, or preventing bone resorption comprising apomegranate extract and at least one of the following natural,plant-derived extracts: Siberian ginseng extract, Ginkgo biloba extract,green tea extract, Sophora japonica extract, Rehmannia sp. extract,grape seed extract, Dong Quai extract, and ipriflavone, wherein thecomposition inhibits expression, production, and/or release of RANK-L,and further wherein, the pomegranate extract is present in an amountranging from approximately 10-2000 mg, more preferably in an amountranging from approximately 300-1700 mg, more preferably in an amountranging from approximately 400-1500 mg, more preferably in an amountranging from approximately 500-1250 mg, more preferably in an amountranging from approximately 600-1000 mg, more preferably in an amountranging from approximately 700-900 mg; more preferably in an amount ofapproximately 500 mg; and if present, Siberian ginseng extract ispresent in an amount ranging from approximately 100-2000 mg, morepreferably in an amount ranging from approximately 300-1700 mg, morepreferably in an amount ranging from approximately 400-1500 mg, morepreferably in an amount ranging from approximately 500-1250 mg, morepreferably in an amount ranging from approximately 600-1000 mg, morepreferably in an amount ranging from approximately 700-900 mg; morepreferably in an amount of approximately 500 mg; Ginkgo biloba extractis present in an amount ranging from approximately 10-1000 mg, morepreferably in an amount ranging from approximately 100-900 mg, morepreferably in an amount ranging from approximately 200-800 mg, morepreferably in an amount ranging from approximately 300-700 mg, morepreferably in an amount ranging from approximately 400-600 mg, morepreferably in an amount of approximately 500 mg; green tea extract ispresent in an amount ranging from approximately 300-700, more preferablyin an amount ranging from approximately 350-650 mg, more preferably inan amount ranging from approximately 400-600 mg, more preferably in anamount of approximately 500 mg; Sophora japonica extract is present inan amount ranging from 10-1000 mg, more preferably in an amount rangingfrom approximately 100-900 mg, more preferably in an amount ranging fromapproximately 200-800 mg, more preferably in an amount ranging fromapproximately 300-700 mg, more preferably in an amount ranging fromapproximately 400-600 mg, more preferably in an amount of approximately500 mg; Rehmannia sp. extract is present in an amount ranging fromapproximately 10-1000 mg, more preferably in an amount ranging fromapproximately 100-900 mg, more preferably in an amount ranging fromapproximately 200-800 mg, more preferably in an amount ranging fromapproximately 300-700 mg, more preferably in an amount ranging fromapproximately 400-600 mg, more preferably in an amount of approximately500 mg; grape seed extract is present in an amount ranging from 35-250mg, more preferably in an amount ranging from approximately 50-150 mg,more preferably in an amount ranging from approximately 75-125 mg; Dongquai extract is present in an amount ranging from approximately 10-1000mg, more preferably in an amount ranging from approximately 100-900 mg,more preferably in an amount ranging from approximately 200-800 mg, morepreferably in an amount ranging from approximately 300-700 mg, morepreferably in an amount ranging from approximately 400-600 mg, morepreferably in an amount of approximately 500 mg; and ipriflavone ispresent in an amount ranging from approximately 400-700 mg, morepreferably in an amount ranging from approximately 450-650 mg, morepreferably in an amount ranging from approximately 500-600 mg, morepreferably in an amount of approximately 600 mg; wherein the combinationinhibits RANK-L expression, production and/or release of RANK-L orinhibits release of calcium from bone.

In a further example, the present invention is a composition forinhibiting, decreasing, or preventing bone resorption comprising acombination of approximately 10-2000 mg of pomegranate extract, morepreferably approximately 400-1700 mg, more preferably approximately500-1500 mg, more preferably approximately 600-1250 mg, more preferablyapproximately 700-1000 mg, more preferably approximately 800-900 mg;approximately 35-250 mg of grape seed extract, more preferablyapproximately 50-150 mg, more preferably approximately 75-125 mg;approximately 400-700 mg of ipriflavone, more preferably approximately450-650 mg, more preferably approximately 500-600 mg, more preferablyapproximately 600 mg; and approximately 300-700 mg of a green teaextract, more preferably approximately 350-650 mg, more preferablyapproximately 400-600 mg, more preferably approximately 500 mg, whereinthe combination inhibits expression, production, and/or release ofRANK-L or inhibits release of calcium from bone.

In another example, the present invention is a composition forinhibiting, decreasing, or preventing bone resorption comprising acombination of approximately 10-2000 mg of pomegranate extract, morepreferably approximately 400-1700 mg, more preferably approximately500-1500 mg, more preferably approximately 600-1250 mg, more preferablyapproximately 700-1000 mg, more preferably approximately 800-900 mg;approximately 35-250 mg of grape seed extract, more preferablyapproximately 50-150 mg, more preferably approximately 75-125 mg; andapproximately 400-700 mg of ipriflavone, more preferably approximately450-650 mg, more preferably approximately 500-600 mg, more preferablyapproximately 600 mg, wherein the combination inhibits expression,production, and/or release of RANK-L or inhibits release of calcium frombone.

In another example, the invention is a composition for inhibiting,decreasing, or preventing bone resorption comprising a combination ofpomegranate extract, grape seed extract, and ipriflavone, wherein thegrape seed extract is present in an amount of approximately 1/10 thepomegranate extract and the ipriflavone is present in an amount ofapproximately 400-700 mg, more preferably approximately 450-650 mg, morepreferably approximately 500-600 mg, more preferably approximately 600mg, wherein the combination inhibits expression, production, and/orrelease of RANK-L or inhibits release of calcium from bone.

In a further example, the invention is a composition for inhibiting,decreasing, or preventing bone resorption comprising a combination of atleast two of pomegranate extract, grape seed extract, ipriflavone, andgreen tea extract, wherein, if present, the pomegranate extract ispresent in an amount ranging from approximately 25-100% w/w of thecomposition, more preferably from approximately 30-90% w/w of thecomposition, more preferably from approximately 40-80% w/w of thecomposition, more preferably from approximately 45-60% w/w of thecomposition, more preferably approximately 50% w/w of the composition;the grape seed extract is present in an amount ranging fromapproximately 1-25% w/w of the composition, more preferablyapproximately 2-15% w/w of the composition, more preferablyapproximately 5-10% w/w of the composition; the Ipriflavone is presentin an amount ranging from approximately 25-100% w/w of the composition,more preferably from approximately 30-90% w/w of the composition, morepreferably from approximately 40-80% w/w of the composition, morepreferably from approximately 45-60% w/w of the composition, morepreferably approximately 50% w/w of the composition; and the green teaextract is present in an amount ranging from approximately 1-25% w/w ofthe composition, more preferably approximately 2-15% w/w of thecomposition, more preferably approximately 5-10% w/w of the composition,further wherein the combination inhibits expression, production, and/orrelease of RANK-L or inhibits release of calcium from bone.

Another example of the invention is a method of inhibiting, decreasing,or preventing bone resorption in a subject comprising administering tothe subject a composition comprising natural, plant-derived extracts,including at least one of the following extracts: pomegranate, Siberianginseng, Ginkgo biloba, green tea, Sophora japonica, Rehmannia sp.,grape seed, Dong Quai, and ipriflavone, wherein the composition inhibitsexpression, production, and/or release of RANK-L or inhibits release ofcalcium from bone.

In a further example, the invention is a method of inhibiting,decreasing, or preventing bone resorption in a subject comprisingadministering to the subject a composition comprising a combination ofapproximately 10-2000 mg of pomegranate extract, more preferablyapproximately 400-1700 mg, more preferably approximately 500-1500 mg,more preferably approximately 600-1250 mg, more preferably approximately700-1000 mg, more preferably approximately 800-900 mg; approximately35-250 mg of grape seed extract, more preferably approximately 50-150mg, more preferably approximately 75-125 mg; approximately 400-700 mg ofipriflavone, more preferably approximately 450-650 mg, more preferablyapproximately 500-600 mg, more preferably approximately 600 mg; andapproximately 300-700 mg of a green tea extract, more preferablyapproximately 350-650 mg, more preferably approximately 400-600 mg, morepreferably approximately 500 mg, wherein the combination inhibitsexpression, production, and/or release of RANK-L or inhibits release ofcalcium from bone.

In a further example, the invention is a method of inhibiting,decreasing, or preventing bone resorption in a subject comprisingadministering to the subject a composition comprising a combination ofapproximately 10-2000 mg of pomegranate extract, more preferablyapproximately 400-1700 mg, more preferably approximately 500-1500 mg,more preferably approximately 600-1250 mg, more preferably approximately700-1000 mg, more preferably approximately 800-900 mg; approximately35-250 mg of grape seed extract, more preferably approximately 50-125mg, more preferably approximately 75-100 mg; and approximately 400-700mg of ipriflavone, more preferably approximately 450-650 mg, morepreferably approximately 500-600 mg, more preferably approximately 600mg, wherein the combination inhibits expression, production, and/orrelease of RANK-L or inhibits release of calcium from bone.

In a further example, the invention is a dietary supplement regimen forincreasing or stimulating bone growth and inhibiting, decreasing, orpreventing bone resorption comprising a first composition comprising acombination of at least two of: quercetin dihydrate, quercetinanhydrate, Siberian ginseng extract, licorice extract, and Sophorajaponica extract, wherein the combination of the first compositionincreases the expression and/or activity of BMP-2; and a secondcomposition comprising a combination of at least two of: pomegranateextract, grape seed extract, ipriflavone, and green tea extract, whereinthe combination of the second composition inhibits the expression ofRANK-L or inhibits the release of calcium from bones.

In a further example, the invention is a dietary supplement regimen forincreasing or stimulating bone growth and inhibiting, decreasing, orpreventing bone resorption comprising a first composition comprising acombination of at least two of: quercetin anhydrate, quercetindihydrate, Siberian ginseng extract, licorice extract, and Sophorajaponica extract, wherein, if present, quercetin anhydrate or quercetindihydrate is present in an amount ranging from approximately 10-1000 mg,more preferably in an amount ranging from approximately 200-750 mg, morepreferably in an amount ranging from approximately 300-700 mg, morepreferably in an amount ranging from approximately 400-600 mg, morepreferably in an amount of approximately 500 mg; wherein, if present,Siberian ginseng extract is present in an amount ranging fromapproximately 100-800 mg, more preferably in an amount ranging fromapproximately 200-750 mg, more preferably in an amount ranging fromapproximately 300-700 mg, more preferably in an amount ranging fromapproximately 400-600 mg, more preferably in an amount of approximately500 mg; wherein, if present, licorice extract is present in an amountranging from 10-500 mg, more preferably in an amount ranging fromapproximately 25-450 mg, more preferably in an amount ranging fromapproximately 50-400 mg, more preferably in an amount ranging fromapproximately 75-350 mg, more preferably in an amount ranging fromapproximately 100-300 mg, more preferably in an amount ranging fromapproximately 125-250 mg, more preferably in an amount ranging fromapproximately 25-175 mg; and wherein if present, Sophora japonicaextract is present in an amount ranging from approximately 10-1000 mg,more preferably in an amount ranging from approximately 100-900 mg, morepreferably in an amount ranging from approximately 200-800 mg, morepreferably in an amount ranging from approximately 300-700 mg, morepreferably in an amount ranging from approximately 400-600 mg, morepreferably in an amount of approximately 500 mg, and further wherein thecombination of the first composition increases the expression and/oractivity of BMP-2; and a second composition comprising a combination ofat least two of: pomegranate extract, grape seed extract, ipriflavone,and green tea extract, wherein, if present, pomegranate extract ispresent in an amount ranging from approximately 10-2000 mg, morepreferably approximately 400-1700 mg, more preferably approximately500-1500 mg, more preferably approximately 600-1250 mg, more preferablyapproximately 700-1000 mg, more preferably approximately 800-900 mg;wherein, if present, grape seed extract is present in an amount rangingfrom approximately 35-250 mg, more preferably approximately 50-150 mg,more preferably approximately 75-125 mg; wherein, if present,ipriflavone is present in an amount ranging from approximately 400-700mg, more preferably approximately 450-650 mg, more preferablyapproximately 500-600 mg, more preferably approximately 600 mg; andwherein, if present, green tea extract is present in an amount rangingfrom approximately 300-700 mg, more preferably approximately 350-650 mg,more preferably approximately 400-600 mg, more preferably approximately500 mg, and further wherein the combination of the second compositioninhibits the expression of RANK-L or inhibits the release of calciumfrom bones.

MODES OF ADMINISTRATION

The compositions of the invention may be administered systemically orlocally. For systemic use, the compositions of the invention areformulated for parenteral (e.g., intravenous, subcutaneous,intramuscular, intraperitoneal, intranasal or transdermal) or enteral(e.g., oral or rectal) delivery according to conventional methods.Intravenous administration can be by a series of injections or bycontinuous infusion over an extended period. Administration by injectionor other routes of discretely spaced administration can be performed atintervals ranging from weekly to once to three times daily.Alternatively, the compositions disclosed herein may be administered ina cyclical manner (administration of disclosed composition; followed byno administration; followed by administration of disclosed composition;and the like). Treatment can continue until the desired outcome isachieved. Alternatively, administration of the compositions of thepresent invention may be continual, and thereby be a preventativeadministration, rather than an administration for treatment.

In general, compositions of the present invention can include acosmetically or pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water, borate-buffered saline containingtrace metals or the like. Compositions of the present invention mayfurther include one or more excipients, for example, vitamin A, vitaminD, or calcium; preservatives; solubilizers; buffering agents; albumin toprevent protein loss on vial surfaces; lubricants; fillers; stabilizers;etc. Methods of formulation are well known in the art and are disclosed,for example, in Remington's Pharmaceutical Sciences, Gennaro, MackPublishing Co., Easton Pa., 1990, which is incorporated herein byreference.

Compositions for use within the present invention can be in the form ofsterile, non-pyrogenic liquid solutions or suspensions, coated capsules,suppositories, lyophilized powders, transdermal patches or other formsknown in the art. Local administration may be by injection at the siteof injury or defect, or by insertion or attachment of a solid carrier atthe site, or by direct, topical application of a viscous liquid, or thelike. For local administration, the delivery vehicle preferably providesa matrix for the growing bone or cartilage, and more preferably is avehicle that can be absorbed by the subject without adverse effects.

Aqueous suspensions may contain the extract ingredients of the presentinvention in admixture with pharmacologically acceptable excipients suchas vitamin A, vitamin D, and calcium, suspending agents, such as methylcellulose; and wetting agents, such as lecithin, lysolecithin orlong-chain fatty alcohols. The said aqueous suspensions may also containpreservatives, coloring agents, flavoring agents, sweetening agents andthe like in accordance with industry standards.

Preferably, compositions of the present invention are orallyadministered in the form of a pill, tablet, powder, bar, food, beverage,lozenge, etc. Additionally, compositions of the present invention may bepresented as a dried or powdered product for reconstitution with wateror other suitable vehicle before use. Liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, cellulosederivatives or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid).

When administered in the form of a beverage, compositions of the presentinvention may be water-based, milk-based, tea-based, fruit juice-based,or some combination thereof.

Compositions of the present invention may also be orally administered inthe form of a solid prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinized maizestarch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The solids may be coatedby methods well-known in the art. In a preferred embodiment, thecomposition of the present invention may take the form of a two-piecehard shell capsule, a soft gelatin capsule, or a powder to be dissolvedin a liquid for oral consumption. Preparations for oral administrationmay be suitably formulated to give controlled release of the activecompound.

Compositions of the present invention that are orally administered canfurther comprise thickeners, including xanthum gum,carboxymethyl-cellulose, carboxyethyl-cellulose,hydroxypropyl-cellulose, methyl-cellulose, microcrystalline cellulose,starches, dextrins, fermented whey, tofu, maltodextrins, polyols,including sugar alcohols (e.g., sorbitol and mannitol), carbohydrates(e.g. lactose), propylene glycol alginate, gellan gum, guar, pectin,tragacanth gum, gum acacia, locust bean gum, gum arabic, gelatin, aswell as mixtures of these thickeners. These thickeners are typicallyincluded in the formulations of the present invention at levels up toabout 0.1%, depending on the particular thickener involved and theviscosity effects desired.

Orally administered compositions of the present invention can, andtypically will, contain an effective amount of one or more sweeteners,including carbohydrate sweeteners and natural and/or artificial no/lowcalorie sweeteners. The amount of the sweetener used in the formulationsof the present invention will vary, but typically depends on the type ofsweetener used and the sweetness intensity desired.

In addition to the formulations described previously, the compounds mayalso be a formulated as a sustained and/or timed release formulation.Common timed and/or controlled release delivery systems include, but arenot be restricted to, starches, osmotic pumps, or gelatin microcapsules.

The compositions may, if desired, be presented in a pack or dispenserdevice which may comprise one or more unit dosage forms comprising acomposition of the present invention. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

Preparations of compositions of the present invention for topical andlocal application comprise aerosol sprays, lotions, gels and ointmentsin cosmetically or pharmaceutically appropriate vehicles which maycomprise lower aliphatic alcohols, polyglycols such as glycerol,polyethylene glycol, esters of fatty acids, oils and fats, andsilicones. The preparations may further comprise antioxidants, such asascorbic acid or tocopherol, and preservatives, such as p-hydroxybenzoicacid esters.

Parenteral preparations comprise particularly sterile or sterilizedproducts.

Injectable compositions may be provided containing a combination of theextracts of the present invention and any of the well known injectablecarriers. These may contain salts for regulating the osmotic pressure.

Other useful dosage forms can be prepared by methods and techniques thatwill be well understood by those of skill in the art and may include theuse of additional ingredients in producing tablets, capsules, or liquiddosage forms. Although exemplary dosages, dose frequencies, and methodsof administration are discussed herein, these are merely exemplary andit is appreciated that the dose, dose frequency, and mode ofadministration may vary according to the age, body weight, condition andresponse of the individual consumer or patient, and the particularcomposition of the present invention that is used.

EXAMPLES

It is not the policy of applicants to engage in animal testing. However,applicants obtained significant results through in vitro testing ofpotential ingredients to be used in compositions and methods of thepresent invention, and although in vitro tests can provide usefulinformation, such tests cannot replace animal studies because bone, asan organ, together with the complex mechanisms controlling it, cannot bemimicked in the laboratory. Therefore, Applicants performed severalanimal studies, described below, to confirm the activity of compositionsand methods useful in the present invention. In performing all animalstudies, Applicants followed the guidance for handling and humanetreatment of animals as outlined, for example, in the Guidelines forPreclinical and Clinical Evaluation of Agents Used in the Prevention orTreatment of Postmenopausal Osteoporosis, published April, 1994 by theFood and Drug Administration, Division of Metabolic and Endocrine DrugProducts, the entire contents of which are incorporated herein byreference. See also Guidelines for Preclinical Evaluation and ClinicalTrials in Osteoporosis published by the World Health Organization in1998 (ISBN number 9789241545228) and Wark, John D. and Westmore, Ann,Studies of drugs and other measures to prevent and treat osteoporosis; aguide for non-experts, available atwww.who.int/entity/ageing/publications/noncommunicable/alc_osteoporosis_brief.pdf—(lastvisited Oct. 21, 2007), the entire contents of which are incorporated byreference herein.

Example 1 Expression/Activity of BMP-2 Promoter, Gene, and Protein

2T3-BMP2-Luciferase cells, which are murine fibroblast cells transfectedwith BMP-2 promoters linked to the reporter gene luciferase, arecultured using alpha-MEM 10% FCS with 1% penicillin/streptomycin and 1%glutamine and are split ⅕ once per week. The cells are plated inmicrotiter plates at a cell density of 5×10³ cells/100 μl/well. Thecells are allowed to adhere and stabilize using a preincubation periodof 24 hrs at 37° C. with 5% CO₂. The media is removed and replaced with50 μl of □alpha-MEM 4% FCS. 50 μl of Serum Free (0.1% BSA) containingthe compound or factor (2×) to be tested is added to each well. Thefinal volume is 100 μl and the final serum concentration is 2% FCS. Aroutine positive control used is recombinant human BMP2 (“rhBMP2”) orChinese Hamster Ovary-BMP2 (“CHO-BMP2”) conditioned media. The treatedcells are then incubated at 37° C., 5% CO₂ for 48 hrs. Media is thenremoved and the cells are rinsed 3 times with PBS. Excess PBS is removedfrom the wells and 100 μl of cell culture lysing reagent (Promega #E153A) is added to each well and incubated for at least 10 min. 10 μl ofthe cell lysate is added to a 96 well white luminometric plate (DynatechLabs #0010107100) containing 100 μl of luciferase assay buffer withsubstrate (Promega # E152A). The luciferase activity is read using aDynatech ML2250 automated 96 well luminometer. The data is thenexpressed as either percentage of luciferase activity/well or percentageof luciferase activity/pg protein.

The following compounds were tested for their ability to activate theBMP-2 promoter: quercetin, Rehmannia sp. extract, Rehmannia sp. rootextract, Siberian ginseng extract, Sophora japonica extract, licoriceextract, ipriflavone, and cal-z-bone. The results and concentrationstested are reported below at Table 1:

TABLE 1 BMP2 Promoter assay (ratio to control) Conc. R-1 R-2 R-3 SFJ SJSG Q I L CZB μg/ml FOLD* 100 1.00 0.90 0.80 2.00 0.80 1.10 5.20 4.501.40 0.90 50 0.90 0.90 0.90 1.60 0.70 0.90 4.80 4.40 1.40 0.90 25 0.901.10 0.95 1.00 0.84 1.30 3.60 3.90 1.00 1.10 12.5 0.90 1.30 0.93 1.000.77 1.20 2.90 3.60 0.80 1.90 6.3 1.00 1.10 0.88 0.90 0.87 1.30 2.302.90 0.80 1.40 3.2 1.10 1.10 0.93 0.90 0.87 1.40 1.80 2.10 0.90 1.10 1.61.00 1.20 0.95 1.40 0.88 1.20 1.30 1.50 0.70 1.30 0.8 1.10 1.10 0.980.90 0.85 1.20 1.15 1.20 0.80 1.20 0.4 0.90 1.00 0.99 0.90 0.85 1.101.00 0.90 0.80 1.20 0.2 1.00 0.90 0.86 0.90 0.79 1.20 1.10 1.00 0.801.10 0.1 0.90 0.90 0.87 0.80 0.82 1.10 1.10 0.90 1.20 1.10 0.05 0.800.90 1.08 1.20 0.84 1.00 1.00 1.00 0.70 1.10

In Table I, R-1=Rehmannia sp. extract (EUL), R-2=Rehmannia sp. extract(Draco), R-3=Rehmannia sp. root (NuPharma), SFJ=Sophora fructusjaponica, SJ=Sophora japonica (NuPharma), SG=Siberian ginseng,Q=Quercetin, I=Ipriflavone, L=Licorice, CZB=Cal-Z-bone. *Note: If thefold value is 2 or greater, the treatment significantly activated BMP-2promoter. If the fold value is less than 2, the treatment had no effecton BMP-2 promoter. Thus, according to this assay, quercetin dihydrateand ipriflavone were the most potent activators of the BMP-2 promoter.

It is also possible to test for BMP-2 gene expression and proteinexpression using assays similar to those described above for testingBMP-2 promoter activity. In particular, for gene expression, the humanosteosarcoma cell line, MG-63 (ATCC# CRL-1427), is maintained inphenol-red containing MEM (as recommended by ATCC) at 37° C. and 5% CO₂.Twenty-four hours prior to experimentation, 3×10⁵ cells are seeded in12-well plates in phenol-red free MEM. For each experiment, cells aretreated with test extracts at treatment concentrations of 10, 1, and 0.1μg/ml. After an overnight incubation, RNA is extracted usingconventional trizol/guanidine isothiocyanate based lysis. The isolatedRNA was digested with RNase-free DNase I to remove any DNA contaminationand then reverse transcribed to cDNA using random hexamer as well asoligo(dT) primers according to the manufacturer's instructions(Stratagene). Quantitative real time PCR is performed using FAM-labeledspecific primers for BMP-2 and HEX-labeled specific primers for 18S rRNA(Invitrogen). All reactions are carried out in triplicate and therelative amount of mRNAs in treated versus untreated samples iscalculated using the comparative C_(T) method established by AppliedBiosystems (2001). Gene expression changes of 2-fold or greater areconsidered significant.

To measure BMP-2 protein expression, Hu09 cells are plated in 96-wellculture plates at the density of 1×10⁴ cells/well and cultured withalpha-MEM supplemented with 10% FCS for 24 hours. The cells are treatedwith different proteasome inhibitors for 24 hours. After incubation, theconditioned media is transferred into microcentrifuge tubes andcentrifuged at 14,000 rpm for 2-3 minutes to remove cellular debris. Theconcentration of BMP-2 protein in the supernatant is then determinedusing the standard BMP-2 Elisa Kit (R&D ELISA KIT DBP200). RecombinanthBMP-2 (R&D) is used as a standard.

The following compounds were tested for their ability to increase theexpression of BMP-2 gene expression and protein expression: quercetin,Rehmannia sp. extract, Rehmannia sp. root extract, Siberian ginsengextract, Sophora japonica extract, licorice extract, ipriflavone, andcal-z-bone. The results and concentrations tested in the proteinexpression assay are reported below in Table 2.

TABLE 2 BMP-2 Protein Expression Assay Conc. R-1 R-2 R-3 SFJ SJ SG Q I LCZB μg/ml FOLD* 100 <1.3 <1.3 <1.3 3.0 <1.3 2.4 2.2 1.8 <1.3 <1.3 10<1.3 <1.3 <1.3 3.0 <1.3 3.4 4.6 1.8 <1.3 <1.3 1 <1.3 <1.3 <1.3 2.2 <1.32.0 2.0 2.4 <1.3 <1.3 0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

In Table 2, R-1=Rehmannia sp. extract (EUL), R-2=Rehmannia sp. extract(Draco), R-3=Rehmannia sp. root (NuPharma), SFJ=Sophora fructusjaponica, SJ=Sophora japonica (NuPharma), SG=Siberian ginseng,Q=Quercetin, I=Ipriflavone, L=Licorice, and CZB=Cal-Z-bone. *Note: Ifthe fold value is 2 or greater, the treatment significantly activatedBMP-2 protein expression. If the fold value is less than 2, thetreatment had no effect on BMP-2 protein expression.

The results and concentrations tested in the gene expression assay arelisted below in Table 3. An increase in gene expression that is a changeof 2-fold or greater is considered significant.

TABLE 3 BMP-2 Gene Expression Assay, 10 μg/ml Test Ingredient: FoldChange in Gene Expression: Rehmannia sp. extract (EUL)   28 × increaseRehmannia sp. extract (Draco) 11.7 × increase Rehmannia sp. root(NuPharma)  6.3 × increase Sophora fructus japonica   49 × increaseSophora japonica (NuPharma)  8.3 × increase Siberian ginseng   27 ×increase Quercetin  2.6 × increase Ipriflavone No effect Licorice   45 ×increase

Additional results of the gene expression assay are listed below inTable 4. In Table 4, Q=quercetin, SG=Siberian ginseng, SJ=Sophorajaponica, and L=licorice. An increase in gene expression that is 2-foldor greater is considered significant. Synergy was found at a 10:5 or 2:1and 10:10 or 1:1 ratio of quercetin to licorice over quercetin alone.

TABLE 4 BMP-2 Gene Expression Assay BMP-2 Expression Ingredient(dosage): N (fold change) Q (100 μg/ml) 1 14 Q (20 μg/ml) 2 4.61 ± 0.07Q (10 μg/ml) 1 5 Q (10 μg/ml) 2 3.48 ± 0.08 Q (10 μg/ml) 2 3.54 ± 0.12 Q(10 μg/ml) 2 3.52 ± 0.12 Q (5 μg/ml) 2 2.51 ± 0.14 Q (1 μg/ml) 1 2 Q (1μg/ml) 1 2.03 Q (0.1 μg/ml) 1 No change (−1.06) SG (10 μg/ml) 2 11.26 ±0.14  SG (10 μg/ml) 2 12.38 ± 1.00  SG (1 μg/ml) 2 5.61 ± 1.45 SG (0.1μg/ml) 2 2.58 ± 0.38 SJ (10 μg/ml) 2 29.10 ± 2.71  SJ (10 μg/ml) 2 32.45± 0.32  SJ (10 μg/ml) 2 32.80 ± 1.12  SJ (5 μg/ml) 2 23.43 ± 0.23  SJ(2.5 μg/ml) 2 14.89 ± 0.95  SJ (1 μg/ml) 2 11.64 ± 2.04  SJ (0.1 μg/ml)2 3.72 ± 0.37 L (100 μg/ml) 1 29 L (10 μg/ml) 1 8 L (1 μg/ml) 1 4 Q + SG(10 μg/ml each) 2 No change (0.07 ± 1.82) Q + SG (10 μg/ml each) 1 Nochange (1.36) Q + SJ (10 μg/ml each) 2 12.01 ± 0.76  Q (10 μg/ml) + SJ(1 μg/ml) 2 7.09 ± 0.75 Q (10 μg/ml) + SJ (0.1 μg/ml) 2 2.63 ± 0.15 Q (1μg/ml) + SJ (10 μg/ml) 2 No change (1.30 ± 0.17) Q (5 μg/ml) + SJ (2.5μg/ml) 2 4.07 ± 0.02 Q (10 μg/ml) + SJ (5 μg/ml) 2 13.86 ± 1.22  Q (20μg/ml) + SJ (10 μg/ml) 2 16.86 ± 0.41  SG (10 μg/ml) + SJ (10 μg/ml) 21.97 ± 0.62 SG (10 μg/ml) + SJ (1 μg/ml) 2 3.36 ± 0.04 SG (10 μg/ml) +SJ (0.1 μg/ml) 2 4.57 ± 0.87 Q (100 μg/ml) + L (100 μg/ml) 1 38 Q (100μg/ml) + L (10 μg/ml) 1 252 Q (100 μg/ml) + L (1 μg/ml) 1 35 Q (10μg/ml) + L (100 μg/ml) 1 92 Q (10 μg/ml) + L (10 μg/ml) 1 71 Q (10μg/ml) + L (1 μg/ml) 1 15 Q (1 μg/ml) + L (100 μg/ml) 1 31 Q (1 μg/ml) +L (10 μg/ml) 1 37 Q (1 μg/ml) + L (1 μg/ml) 1 6 Q + SG + SJ (10 μg/mleach) 2 No change (1.39 ± 0.26) Q (0 μg/ml) 3 1.01 ± 0.05 Q (10 μg/ml) 37.22 ± 0.51 L (0 μg/ml) 3 1.01 ± 0.05 L (1 μg/ml) 3 1.34 ± 0.12 L (5μg/ml) 3 0.81 ± 0.03 L (10 μg/ml) 3 1.04 ± 0.04 Q (10 μg/ml) + L (1μg/ml) 3 8.54 ± 2.19 Q (10 μg/ml) + L (5 μg/ml) 3 13.50 ± 4.49  Q (10μg/ml) + L (10 μg/ml) 3 14.77 ± 3.04 

Example 2 Calvarial Assay to Measure Bone Growth

Neonatal murine calvaria from 4 day old Swiss white mice are excised,placed into BGJ media (Sigma), and cut in half along the midline suture.One milliliter of BGJ media supplemented with 0.1% bovine serum albumin(BSA) is added to each well of a 12 well tissue culture plate (Costar)to which the relevant factors or vehicles are added. Each half calvariais placed on a stainless steel grid in the above media at the media/airinterface. Usually 4 bones per experimental group are used. RecombinantBMP-2 (5 μg/ml), FGF (100 ng/ml) or insulin (10 μg/ml) can be used as apositive stimulator of bone formation. Factors to be tested are added onDay 0 (the day of dissection) and media is changed at 24 and 96 hours(if a 7 day assay is required). Calvaria are maintained in humidifiedair (5% Co2), at 37° C. Following the period of incubation, the calvariaare then removed and fixed overnight in 10% formalin. The calvaria arethen decalcified in 14% EDTA overnight and are then embedded in paraffinwax. Sections are then cut, using a standard microtome, along themidline suture to reveal the coronal suture and the region posterior tothis suture. Four micrometer sections were taken initially and at twosequential 400 nm depths. Sections are placed on coated glass slides(Superfrost plus, Fisher Scientific, Pittsburgh, Pa.) and stained withhematoxylin and eosin. The total and new bone area (expressed as mm²),suture thickness (mm), and number of cells lining the bone surface aredetermined in the section posterior to the sagittal suture.Histomorphometric analysis is performed using the Osteomeasure System(Osteometrics INC, Atlanta, Ga.).

The extracts tested in this assay include Sophora fructus japonicaextract, Siberian ginseng extract, quercetin dihydrate, quercetinanhydrate, ipriflavone, licorice, and Cal-Z-bone. The results of testingthese extracts in the calvarial assay are reported below in Tables 5 and6. In Table 5, SJ=Sophora fructus japonica, SG=Siberian Ginseng,Q=Quercetin, I=Ipriflavone, L=Licorice, and CZB=Cal-Z-Bone. BMP-2 andSimvastatin were used as positive controls. In the below table,superscript a (^(a)) means that the result is significantly different(p<0.05) to the blank sample (concentration at 0 μg/ml). Differenceswith p≦0.05 (i.e. where the margin of error is less than or equal to 5%)are considered significant. The first four lines of data on Table 5 arebased on mixtures of various ratios of the ingredients, e.g. 1:1 under“Q:SG” indicates that the quercetin dihydrate and Siberian ginsengingredients are present in that mixture at the same amounts.

TABLE 5 Calvarial Assay Results- Bone formation Q:SG Q:SJ Q:SG:SJ Q:LQ:SJ:L Q:SG:L Q:SG:SJ:L Conc 1:1 10:1 10:10:1 10:1 10:01:1 10:10:110:5:1:1 μg/ml mm² × 10⁻³ 100 0.10 ± 0.12 0.10 ± 0.15 0.10 ± 1.20 0.10 ±0.15 0.10 ± 0.15 0.10 ± 0.15 0.10 ± 0.15 10 11.40 ± 1.15^(a) 0.10 ± 0.1512.70 ± 0.86^(a) 8.22 ± 2.09 3.52 ± 0.75 13.10 ± 1.18^(a) 13.80 ±2.49^(a) 1 13.00 ± 1.30^(a) 7.16 ± 1.39 12.10 ± 1.71^(a) 10.20 ± 3.5212.50 ± 1.60^(a) 11.70 ± 1.28^(a) 9.85 ± 1.51 0 5.65 ± 0.36 5.65 ± 0.365.65 ± 0.36 6.73 ± 0.58 6.73 ± 0.58 6.73 ± 0.58 6.51 ± 0.58 Conc. SFJ SGQ I L CZB μg/ml Mm² × 10⁻³ 100 6.19 ± 0.74 5.42 ± 0.54^(a) 13.55 ±1.47^(a) 7.04 ± 0.69^(a) 7.31 ± 0.68 6.12 ± 0.92 10 7.48 ± 0.92^(a) 5.43± 1.03^(a) 13.41 ± 2.50^(a) 6.05 ± 1.12 5.99 ± 2.05 6.02 ± 0.774 1 7.02± 0.98 4.80 ± 0.83^(a) 7.57 ± 1.52 2.44 ± 0.66 9.08 ± 0.89^(a) 8.84 ±1.35^(a) 0 4.13 ± 0.62 1.90 ± 0.43 3.93 ± 0.80 3.74 ± 0.42 3.83 ± 0.423.73 ± 0.42 Conc. BMP2 Conc. Simvastatin μg/ml mm² × 10⁻³ μM mm² × 10⁻³50 8.50 ± 1.20 1 12.5 ± 1.00 0.5 0.5 9.50 ± 1.00 0.25 0.25 7.45 ± 1.34 05.90 ± 1.20 0 5.90 ± 0.60

Significantly, the above results indicate that the synergy betweenquercetin and Siberian ginseng extract is more than additive.Specifically, assuming an additive effect, at 1 μg/ml new bone formationof approximately 12 mm²×10 mm⁻³ would be expected. Therefore, at fiftypercent concentration of quercetin and Siberian ginseng extract, theexpected result would be approximately 6 mm²×10⁻³ but the above resultsshow that the combination of quercetin and Siberian ginseng extractachieved new bone formation of approximately 13 mm²×10⁻³.

The calvarial assay described above was repeated with variouscombinations of the extracts useful in compositions of the presentinvention. For Table 6, Q=quercetin, SG=Siberian ginseng, L=licorice. Inthe below table, superscript a (^(a)) means that the result issignificantly different (p<0.05) to the blank sample (concentration at 0μg/ml). Differences with p<0.05 are considered significant. The resultsfor each formulation are reported below in Table 6.

TABLE 6 Calvarial Assay Results - Bone Formation Stimulated by ExtractCombinations Q:SG Q:SG Q:L Q:L Conc. Q Conc. SG Conc. L Conc. 2.5:1 5:12.5:1 5:1 ug/ml mm² × 10⁻³ ug/ml mm² × 10⁻³ ug/ml mm² × 10⁻³ ug/ml Mm² ×10⁻³ 14.3  0.10 ± 0.12 5.7 7.90 ± 0.54^(a) 10  9.80 ± 0.45^(a) 20 0.10 ±0.12 0.10 ± 0.12 0.10 ± 0.12 0.10 ± 0.12  8.3  0.10 ± 0.10 1.7 9.56 ±0.67^(a)  5.7 10.45 ± 0.65^(a) 10 0.10 ± 0.10 0.10 ± 0.10 0.50 ± 0.320.10 ± 0.10  0.7 10.30 ± 0.78^(a) 0.3 8.60 ± 0.78^(a)  1.7 11.56 ±0.80^(a)  1 7.80 ± 0.67^(a) 9.60 ± 0.89^(a) 8.32 ± 0.87^(a) 6.40 ± 0.78 0  4.40 ± 0.52 0 4.40 ± 0.52  0.3  8.60 ± 0.67^(a)  0 4.80 ± 0.32 4.80± 0.12 5.20 ± 0.25 5.20 ± 0.25  0  4.20 ± 0.45 Q + L Q + L Q + L Q + L Qat constant, Q at constant, L at constant, L at constant, Conc. 1 ug/mlConc. 0.2 ug/ml Conc. 2 ug/ml Conc. 2 ug/ml ug/ml mm² × 10⁻³ ug/ml mm² ×10⁻³ ug/ml mm² × 10⁻³ Ug/ml mm² × 10⁻³ Q-1,  6.59 ± 1.54 Q-0.2, L-210.10 ± 1.52^(a) L-2, 15.30 ± 2.41^(a) L-0.2, Q-1 12.60 ± 1.70^(a) L-2Q-1 Q-1, 10.30 ± 2.00 Q-0.2, L-0.5  6.41 ± 1.11 L-2, 13.90 ± 0.42^(a)L-0.2, Q-0.2 13.20 ± 0.77^(a) L-0.5 Q-0.2 Q-1, 12.80 ± 4.42 Q-0.2,  7.04± 1.31 L-2, 11.10 ± 0.38^(a) L-0.2, 10.30 ± 1.27^(a) L-0.125 L-0.125Q-0.625 Q-0.625 Q-1 13.70 ± 3.20^(a) Q-0.2 ug/ml  6.90 ± 0.56 L-2 ug/ml 9.10 ± 0.45^(a) L-0.2 ug/ml  6.80 ± 0.76 Q-0 ug/ml  6.23 ± 0.84 Q-0ug/ml  6.15 ± 0.68 L-0 ug/ml  6.27 ± 0.34 L-0 ug/ml  6.54 ± 0.65

Example 3 In Vivo Study to Measure Bone Growth

Three different formulas were tested in an in vivo study involvingfemale, intact rats, aged approximately 12-14 weeks, with approximatebody weights between 200-250 grams. Formula 1 included dosages ofquercetin anhydrate and licorice at 250 mg quercetin:250 mg licoriceethanol extract and 500 mg quercetin: 500 mg licorice ethanol extract.Formula 2 included dosages of quercetin anhydrate and licorice at 250 mgquercetin: 125 mg licorice ethanol extract; 500 mg quercetin:250 mglicorice ethanol extract; and 1000 mg quercetin:500 mg licorice ethanolextract. Formula 3 included dosages of quercetin and licorice at 1000 mgquercetin:200 mg licorice ethanol extract. The study lasted 35 days.Eight separate groups of rats were tested with 15 rats in each group.Two of the eight groups of rats tested were control groups. One was aplacebo control group and the other group received 50 μg/kg (10 μg/200g)/3×per week parathyroid hormone. Parathyroid hormone, known as ananabolic agent, served as a positive control. See, e.g., Turner et al.,“Disuse in adult male rats attenuates the bone anabolic response to atherapeutic dose of parathyroid hormone.” J. Appl Physiol. 2006. Vol.101:881-886, the entire contents of which are incorporated herein byreference.

Table 7 below indicates the formula given to each of the 8 test groups.The dosages in Table 7 are based on the recommended human daily dosages.FDA dose conversion formula (Human equivalent dose (HED, mg/kg)=rat dosein mg/kg×(rat weight in kg/human weight in kg)^(0.33)) was used toconvert the human dose to the rat dose.

HED=ratNOAEL×(W-rat/W-human)^((1-b)) ,b=0.67.

In Table 7, Q=quercetin and L=licorice.

TABLE 7 Formulas tested in the In Vivo Study Group #: Formula: HumanDaily Dosage (mg): 1 Q + L (Formula 1) - 1:1 Q = 250, L = 250 2 Q + L(Formula 1) - 1:1 Q = 500, L = 500 3 Q + L (Formula 2) - 2:1 Q = 250, L= 125 4 Q + L (Formula 2) - 2:1 Q = 500, L = 250 5 Q + L (Formula 2) -2:1 Q = 1000, L = 500 6 Q + L (Formula 3) - 5:1 Q = 1000, L = 200 7Placebo (null treatment) 8 Positive Control (Parathyroid Hormone - 50μg/kg (10 μg/200 g)/3 × per week)

Female, Sprague Dawley rats were used (200-250 gm each, ˜12-14 weeksold). Animals were fed at 15 gm/day, which included either a placebodiet or one of the formulas listed above, for a period of 35 days fromthe beginning of the experiment. Chow was made available each morning tothe rats at the rate of 15 grams/rat/day. The rats were allowed freeaccess to water and housed in appropriate cages for the entireexperiment. Unrestricted activity was allowed during the entireexperiment, which lasted 35 days.

The effectiveness of the formulas on bone formation was assessed byoutcome measurements that included biomechanical measurement (to assessbone strength and function information), micro-CT (to analyze virtualbone quality and function information), and histomorphology measurement(to analyze bone quality and functional information). Method ofconducting a histomorphological measurement are described by Pa Revell,“Histomorphometry of Bone,” J. Cline. Pathol., 1983. Vol. 36:1321-1331,the entire contents of which are incorporated herein by reference. Theterminology and units used are those recommended by the HistomorphometryNomenclature Committee of the American Society for Bone and MineralResearch. Methods of conducting a micro-CT measurement are described atJiang et al., “Micro CT and Micro MR imagining of 3D architecture ofanimal skeleton.” J. Musculoskel Neuron Interact. 2000. Vol. 1:45-51,the entire contents of which are incorporated herein by reference. Thesemeasurements were determined after 35 days of treatment.

Six animals from each group were sacrificed on day 35 followingtreatment. Femurs were used for histomorphology analysis. To determinethe effect of the different formulas on bone formation, trabecula bonevolume, trabecular cells number and trabecular separation were assessedby histomorphology technique. In the below table, Table 8, superscript a(^(a)) means that the result is significantly different (p<0.05) to theplacebo group (null treatment). Difference with p<0.05 are consideredsignificant. As shown below in Table 8, the positive control group, PTH,showed a significant increase in bone volume (55.83%), a significantincrease in trabecular cells number (19.57%), and a reduction intrabecular separation (38.30%) after treatment with PTH as compared tothe placebo group. Similarly, the formula administered to Group 3demonstrated a significant increase in bone volume (47.59%), asignificant increase in trabecular cells number (24.19%), and areduction in trabecular separation (23.52%) as compared to the placebogroup, and its effectiveness was comparable to the PTH treatment.

TABLE 8 Histomorphological Measurements Group % Bone TrabecularTrabecular Trabecular Bone mineral Bone # volume number Separationthickness apposition rate formation rates 1    3.01%    7.42%  −8.18% −6.83%  −0.23%  4.14% 2  −6.44%    2.06%  −8.51% −11.47%  −2.81% 15.82%3   47.59%^(a)   24.19%^(a) −23.52%   13.04%   26.60%^(a) 51.37%^(a) 4  11.23%    4.71%  −7.26%  −6.14%    9.19% 16.61% 5   29.01%^(a)   4.96%  −9.07%  −8.95%    3.18% 20.28% 6  −6.63%  −8.33%  −7.35%   5.36%    9.59%  3.20% PTH   55.83%^(a)   19.57%^(a) −38.30%    7.35%  28.51%^(a) 45.83%^(a)

Table 8 demonstrates that formulas administered to group 5 demonstrateda significant difference in trabecular bone volume as compared toplacebo (P<0.05).

In addition, Table 8 reports measurements of trabecular bone thickness.A higher value of bone thickness indicates a greater amount of mineraldepositing in the bone, which can result in an increase in bone mass.The formula administered to group 3 significantly increased trabecularbone thickness (13.04%) as compared to the placebo group and itseffectiveness was comparable to PTH treatment (7.35%).

Bone mineral apposition rate (MARs), also reported by Table 8, wasdetermined by measuring the mean interlabel distance divided by the timeinterval (5 days) between the two flurorchromes administered. A greaterbone mineral apposition rate (or mineralizing surface) means that thereis more new bone deposited. This outcome should correspond to trabecularbone thickness outcome. Indeed, after 35 days of treatment, the formulaadministered to group 3 showed a significant increase in bone mineralapposition rate (26.60%) as compared to the placebo groups and itseffectiveness was comparable to PTH treatment (28.51%).

Table 8 additionally provides values for bone formation rates (BFRs).BFRs were calculated from the extent of bone surface labeled withtetracycline (viewed using epifluorescence) and the distance between thelabels in areas where 2 labels are present (calcein and tetracycline)and it was expressed as square micrometer per cubic micrometer per day(μm²/mm³/day). A greater bone formation rate means that more new bonetissues can be formed. The outcome of bone formation rate was consistentwith the observed change of bone mineral apposition rate. The formulaadministered to group 3 showed a significant increase in bone formationrate (51.37%) as compared to the placebo group and its effectiveness wascomparable to PTH treatment (45.83%).

Another efficacy determination technique used in this study was micro-CTmeasurement, which can provide virtual bone quality, structure andfunctional information. The results of this analysis are reported belowin Table 9.

Obtaining information from a wide range of parameters such as bonemineral density (BMD), bone volume (% bone volume/total volume),trabecular number, and trabecular separation is critical for bonestructure assessment quantitatively and qualitatively. Among theseparameters, the outcome of bone volume, trabecular number, andtrabecular separation correlate with bone health.

The results reported in Table 9 indicate that the formulas administeredto groups 2 and 5 effectively enhanced osteoblastogenesis andsignificantly increased bone mineral density (BMD) as compared to theplacebo group and their effectiveness was comparable to PTH treatment.Results also indicate that the formulas administered to groups 1, 2, and5 significantly increased bone volume and the number of trabecular cellsand reduced trabecular separation as compared to the placebo group andtheir effectiveness was comparable to PTH treatment. In Table 9,superscript a (^(a)) indicates the result is significantly different(p<0.05) from the placebo group. Difference with p<0.05 are consideredsignificant.

TABLE 9 micro-ct Measurements % Bone Trabecular Trabecular Bone MineralVolume/ Number separation Group # Density (BMD) Total Volume (N/6 mm²)(mm) 1 17% 32% ^(a) 15% ^(a) −22% ^(a) 2 71% ^(a) 33% ^(a) 14% ^(a) −22%^(a) 3 20% 15%  7%  −9% 4 31% 13%  5%  −8% 5 74% ^(a) 25% ^(a) 14% ^(a)−19% ^(a) 6 20%  9%  4%  −4% PTH 86% ^(a) 32% ^(a) 25% ^(a) −28% ^(a)

Besides bone mass and bone volume evaluation, bone strength was examinedmechanically using two different parameters. One parameter measured themaximum force needed to fracture the bone and the other determined thestiffness (the elasticity) of bone. Methods of determining bone strengthand stiffness are described by Sturmer et al., “Standardized Bending andBreaking Test for the Normal and Osteoporotic Metaphyseal Tibias of theRat: Effect of Estradiol, Testosterone, and Raloxifene,” J. Bone andMineral Research, 2006. Vol. 21(1):89-96, the entire contents of whichare incorporated herein by reference.

In Table 10, results indicate that there were no statistical differencesin the maximum force needed to fracture the bone and stiffness of bonebetween any treatment groups including the positive control group (PTH)and the placebo group. No effect from the positive control group couldbe due to a relative low dose of PTH employed at only a frequency of 3times per week. Although PTH is a powerful anabolic agent, the PTH dosewas specifically chosen to provide a moderate effect on bone formationwithout inducing any adverse effect.

TABLE 10 Biomechanical Measurements Maximum Force Required Group # toFracture Bones Bone Stiffness 1 −0.1% −0.1% 2 −0.1%   0.0% 3   0.0%  0.0% 4   0.0%   0.0% 5 −0.1% −0.1% 6   0.0%   0.0% PTH   0.0%   0.0%

Example 4 Inhibition of RANK-L Study

Weigh between 150 to 250 mg of each ingredient into a 15 ml conicalbottomed tube. Dissolve in a solution of 50% DMSO:30% Ethanol:20% water,such as to end with a final stock solution of 50 μg/ml. For solventcontrol, mix 1.5 ml DMSO, 0.9 ml ethanol, and 0.6 ml water. Vortexthoroughly. Sonicate for 10 minute in water bath, room temperature.Vortex thoroughly again. Dilute ingredient stocks in fresh phenolred-free media.

Human osteosarcoma cell line, MG-63 (ATCC# CRL-1427), is maintained inphenol-red containing MEM (as recommended by ATCC) at 37° C. and 5% CO₂.Twenty-four hours prior to experimentation, 3×10⁵ cells are seeded in12-well plates in phenol-red free MEM. Spent medium is removed fromwells and MG-63 cells (ATCC # CRL-1427) are pretreated with 1, 0.1, 0.01μg/ml of each test ingredient or 30 ng/ml TGF-beta (+control) for 4hours at 37° C., 5% CO₂. Stock solution of TGF-beta is 30 μg/ml (fromR&D Systems, cat#100-B-001).

After pretreatment incubation period, 10 μg/ml IL-1b (Calbiochem(catalog #407615) and stock solution at 10 μg/ml, cat #407615) wereadded to the cell cultures for 18 hours at 37° C. Supernatant media fromthe treated and stimulated cells were removed and total RANK-L proteinswere measured using RANK-L ELISA (in triplicate) as described bymanufacturer (from APOTECH, catalog # APO-54N-016-k101). The resultingprotein quantity was compared to that of media (null) treated stimulatedcells to determine percent decrease in total RANK-L protein.

The above-described assay procedure was used to determine the ability ofextracts of: Ginkgo biloba, green tea, Sophora fructus japonica,Rehmannia sp., pomegranate, Siberian ginseng, ipriflavone, grape seed,Dong quai, and Sophora japonica, to inhibit RANK-L expression,production, or release. The results of this assay are reported below inTable 11. Decreases of 10% or greater are considered significant.

TABLE 11 Production/Release of RANK-L Protein (relative to untreatedcontrol) % Change in Production/Release Levels Ingredient (tested at 1μg/ml) (compared to untreated control) Ginkgo biloba 31% decrease Greentea 19% decrease Sophora Fructus Japonica 45% decrease Rehmannia sp. 74%decrease Pomegranate 20% decrease Pomegranate (Naturex) 14% decreaseSiberian ginseng 50% decrease Ipriflavone No effect Grape Seed Extract11% decrease Dong Quai (20:1 extraction ratio) 16% decrease Sophorajaponica (NuPharma) 42% decrease

Based on the results reported at Table 11, it was determined thatpomegranate extract, ipriflavone (Ostivone), grape seed extract (40%proanthocyanidins) and green tea extract (40% EGCG) showed the positiveeffect on inhibition of RANK-L production/release. Therefore, variouscombinations of these ingredients were tested, using the RANK-Linhibition assay described above, to determine what level of inhibitionof RANK-L production/release could be achieved. The results are reportedbelow in Table 12. As these reports demonstrate, the combination of 10μg/ml of Pomegranate, 10 μg/ml Ipriflavone, 1 μg/ml Grape Seed extract,and 1 μg/ml Green tea was found to be the formula that maximized theinhibition of RANK-L synthesis in response to IL-1B protein stimulus ofosteoblast cells. The results also show that anything with pomegranategenerally performed well. Further, no major interference was found amongthe ingredients.

TABLE 12 RANK-L Inhibition RANK-L inhibition Ingredient (dosage): n (%)Organic Olive Juice Powder (100 μg/ml) 2 41.5 ± 4.0  Organic Olive JuicePowder (10 μg/ml) 6 19.7 ± 2.2  Organic Olive Juice Powder (1 μg/ml) 6No inhibition Organic Olive Juice Powder (0.1 μg/ml) 3 No inhibitionPomegranate extract (30 μg/ml) 2 58.6 ± 1.5  Pomegranate extract (20μg/ml) 2 56.2 ± 2.6  Pomegranate extract (10 μg/ml) 3 48.3 ± 3.1 Pomegranate extract (10 μg/ml) 2 46.9 ± 10.7 Pomegranate extract (10μg/ml) 2 48.4 ± 1.1  Pomegranate extract (10 μg/ml) 3 49.4 ± 3.3 Pomegranate extract (1 μg/ml) 3 19.3 ± 2.1  Pomegranate extract (0.1μg/ml) 3 4.7 ± 4.2 Ipriflavone (Ostivone) (10 μg/ml) 3   0 ± 4.3Ipriflavone (Ostivone) (10 μg/ml) 3 2.8 ± 3.9 Ipriflavone (Ostivone) (10μg/ml) 3   0 ± 2.5 Ipriflavone (Ostivone) (1 μg/ml) 2 0.8 ± 0.3 GrapeSeed Extract (40% OPC) (10 μg/ml) 3 18.7 ± 6.9  Grape Seed Extract (40%OPC) (10 μg/ml) 2 21.9 ± 0.6  Grape Seed Extract (40% OPC) (5 μg/ml) 313.1 ± 3.4  Grape Seed Extract (40% OPC) (2 μg/ml) 2 10.3 ± 1.6  GrapeSeed Extract (40% OPC) (1 μg/ml) 3 8.0 ± 2.2 Grape Seed Extract (40%OPC) (1 μg/ml) 2 10.7 ± 1.0  Grape Seed Extract (40% OPC) (0.1 μg/ml) 2  0 ± 4.3 Green Tea Extract (40% EGCG) (10 μg/ml) 3 23.9 ± 1.4  GreenTea Extract (40% EGCG) (10 μg/ml) 3 22.6 ± 7.5  Green Tea Extract (40%EGCG) (1 μg/ml) 3 10.2 ± 1.6  Green Tea Extract (40% EGCG) (0.1 μg/ml) 3  0 ± 2.6 Pomegranate + Ipriflavone (Ostivone) (10 μg/ml each) 2 45.7 ±2.4  Pomegranate + Ipriflavone (Ostivone) (10 μg/ml each) 2 48.0 ± 2.8 Pomegranate + Ipriflavone (Ostivone) (10 μg/ml each) 3 46.9 ± 5.3 Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (1 μg/ml) 2 48.3 ± 3.5 Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (0.1 μg/ml) 2 45.6 ±04.0 Pomegranate (1 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) 3 21.6 ±5.2  Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) + Grape2 63.3 ± 0.7  Seed Extract (10 μg/ml) Pomegranate (10 μg/ml) +Ipriflavone (Ostivone) (10 μg/ml) + 2 64.6 ± 4.9  Grape Seed Extract (1μg/ml) Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) + 249.9 ± 2.5  Grape Seed Extract (0.1 μg/ml) Pomegranate + Ipriflavone(Ostivone) + Grape Seed Extract 2 42.8 ± 8.9  (10 μg/ml each)Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) + 3 39.0 ±2.9  Green Tea Extract (10 μg/ml) Pomegranate (10 μg/ml) + Ipriflavone(Ostivone) (10 μg/ml) + 2 54.6 ± 1.1  Green Tea Extract (1 μg/ml)Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) + 2 37.5 ±8.1  Green Tea Extract (0.1 μg/ml) Pomegranate + Ipriflavone(Ostivone) + Green Tea Extract 3 23.5 ± 2.3  (10 μg/ml each) Pomegranate(10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) + 3 42.9 ± 1.1  GrapeSeed Extract (10 μg/ml) + Green Tea Extract (10 μg/ml) Pomegranate (10μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) + 2 60.8 ± 0.9  Grape SeedExtract (10 μg/ml) + Green Tea Extract (0.1 μg/ml) Pomegranate (10μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) + 3 55.1 ± 1.8  Grape SeedExtract (1 μg/ml) + Green Tea Extract (0.1 μg/ml) Pomegranate (10μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) + 3 68.6 ± 2.6  Grape SeedExtract (1 μg/ml) + Green Tea Extract (1 μg/ml) Pomegranate (10 μg/ml) +Ipriflavone (Ostivone) (10 μg/ml) + 3 65.2 ± 1.1  Grape Seed Extract (1μg/ml) + Green Tea Extract (10 μg/ml) Pomegranate (10 μg/ml) +Ipriflavone (Ostivone) (10 μg/ml) + 3 52.3 ± 0.7  Grape Seed Extract(0.1) + Green Tea Extract (1 μg/ml) Pomegranate + Ipriflavone(Ostivone) + Grape Seed Extract + 3 38.0 ± 3.5  Green Tea Extract (10μg/ml each) Ipriflavone (Ostivone) + Grape Seed Extract + Green TeaExtract 3 9.7 ± 2.8 (10 μg/ml each) Pomegranate + Grape Seed Extract +Green Tea Extract 3 26.9 ± 4.8  (10 μg/ml each) Pomegranate (10 μg/ml) +Grape Seed Extract (1 μg/ml) 2 52.7 ± 1.0  Pomegranate (10 μg/ml) +Grape Seed Extract (1 μg/ml) 2 51.1 ± 1.8  Pomegranate (20 μg/ml) +Grape Seed Extract (2 μg/ml) 2 58.2 ± 0.5  Pomegranate (30 μg/ml) +Grape Seed Extract (1 μg/ml) 2 64.3 ± 2.1  Pomegranate (30 μg/ml) +Grape Seed Extract (2 μg/ml) 2 59.9 ± 1.2  Pomegranate (30 μg/ml) +Grape Seed Extract (5 μg/ml) 2 57.2 ± 3.1 

Example 5 Isolation of Punicalagins from Pomegranate

Fresh pomegranates were peeled to separate the seeds from the peels. Theseeds, peels, and fruit flesh were separately extracted using a waterand alcohol combination (80:20). Each of the peels, seeds, and fleshyielded extracts with punicalagins but the pomegranate peel extractyielded the highest levels of punicalagins. In addition, water was shownto be the best extraction solvent for extracting puncalagins.

Example 6 Inhibition of RANK-L by Punicalagins

Punicalagin test samples are extracted from pomegranates, including frompomegranate peels, skins, fleshy fruit, and seeds using 100% DMSO at 100mg/ml. Particulate matter in the extraction is not removed. Thepunicalagin test samples are diluted in MEM without phenol red(estrogenic) and 0.1% FBS to lower background signaling to ten times thefinal concentrations. Final DMSO concentrations are kept below 0.2% andare kept constant during treatments. DMSO solvent control is used in theuntreated group.

MG63 cells (ATCC # CRL-1472), a human-derived osteosarcoma cell line,are plated at 200,000 cells per well in a 12 well plate with phenol redfree MEM at 10% FBS. The next day the media is changed to phenol redfree MEM at 0.1% FBS. The cells are incubated for four hours and thentreated with punicalagin test samples at concentrations of 1, 10, and100 μg/ml.

After four hours of treatment, IL-1b (Calbiochem (catalog #407615),stock solution at 10 μg/ml, cat #407615) are added to a finalconcentration of 3 ng/mL. The treated cells the stimulus, IL-1 b, arethen allowed to incubate for 16 hours at 37° C.

After 16 hours the cells are lysed and the RNA is extracted from thecells with the RNasy purification kit (Qiagen). RNA is reversetranscribed to cDNA and quantified by qPCR using 2-step qRT-PCR reagents(Invitrogen), and 4 μL of purified RNA. Annealing temperature is 57° C.in the Stratagene Mx4000 with 1 μL of RANK-L gene specific primers in a50 μL reaction (10 μM initial concentration; HLUX3013920, InvitrogenInc.).

RANK-L and GAPDH gene expression Ct data are obtained. RNA is quantifiedwith adjustments for GAPDH expression. The untreated control values forRANK-L expression are used to evaluate treatment affects. The resultsare reported below in Table 13.

TABLE 13 Effect of Punicalagins on RANK-L Expression Ingredient N RANK-LExpression (fold increase) Untreated 2 1.0 ± 0.1 IL1B (3 μg/ml) 2 3.3 ±0   Punicalagins 1 μg/ml 2 2.6 ± 0.5 Punicalagins 10 μg/ml 2 1.9 ± 0.4Punicalagins 100 μg/ml 2 0.7 ± .02

As shown in Table 13, punicalagins purified from pomegranates inhibitIL-1b stimulated RANK-L gene expression in a dose dependent manner andcompletely inhibit RANK-L expression at 100 μg/mL.

Example 7 Inhibition of Type IV Collagenase (MMP9) Protein Expression byPunicalagins from Pomegranate

Keratinocytes and fibroblasts were co-cultured in DMEM containing 0.5%BSA. Co-cultures were exposed to various concentrations of punicalagins(ranging from 0.1%-10%) were extracted from pomegranates. Specifically,the ability of the pomegranate extracts to inhibit type IV collagenaseprotein (matrix metalloproteinase—9/MMP9) expression at concentrationsof 1.0 μg/ml, 10 μg/ml, and 100 μg/ml were tested. Following exposure tothe pomegranate extracts, the co-culture cells were stimulated with 10ng/ml of IL-1B for 18 hours. Following stimulation with IL-1B for 18hours, and MMP9 concentration was determined in the media as shown belowin Table 14.

TABLE 14 Effect of Pomegranate Extracts on MMP9 (type IV collagenase)protein expression Tested Pomegranate Extract N Inhibition of MMP9Untreated 2 1.0 ± 0.1 IL1B (10 ng/ml) 2 1.64 Punicalagins 1 μg/ml 4 1.5± .06 Punicalagins 10 μg/ml 4 1.2 ± .04 Punicalagins 100 μg/ml 4 1.0 ±0.1

The results reported at Table 14 demonstrate that punicalagins frompomegranate inhibit IL-1B stimulated collagenase release (MMP9) fromkeratinocytes in vitro. These results demonstrate that punicalaginsinhibit inflammation-stimulated breakdown of the extracellular matrix.Activated osteoclasts reduce bone strength and increase bone loss bysecreting matrix digesting enzymes (MMPs) to break down the bone'scollagen/calcium phosphate framework. Blocking the destruction of bone'scollagen/calcium framework will be expected to maintain/improve bonestrength and bone structure. Increased bone strength and bone structureare characterized by increased bone mineral density, increased bonevolume, increased trabecular cell number, decreased trabecularseparation, improved bone architecture, increase in maximum force neededto fracture bone, and increase in stiffness of bone.

Example 8 Inhibition of C. histolyticum Collagenase Activity by GrapeSeed and Pomegranate Extracts

Samples are prepared by weighing out 100 mg of powder. A 50 mg/ml totalextract of the sample is then prepared by sequential addition ofDMSO:Ethanol:water in a ratio of 5:3:2. Therefore for 100 mg of powder,1 ml DMSO, 0.6 ml ethanol, and 0.4 ml water would be used. The solutionsare extensively mixed by vortexing and are then incubated for 10 min ina sonic water bath. The samples are diluted from the stock concentrationof 50 mg/ml to test concentrations.

Inhibition of collagenase activity is assayed using a commerciallyavailable kit (Molecular Probes, Eugene, Oreg.). The kit is based on anability to digest a collagen substrate labeled with a fluorescent tag.Prior to digestion, the fluorescence of the substrate is quenched. Afterexposure to collagenase, the substrate is cleaved abolishing thequenching effect so that the fluorescence increases. The samples(prepared according to above procedure) are first added to thecollagenase (0.2 Units/ml) provided with the kits. The fluorescentsubstrate (50 μg/ml) is then added and the reaction is incubated for anhour at ambient temperature. Fluorescence is read on a plate reader atexcitation/emission of 495/515 nm. Data are expressed as % controlcompared to MMP without any inhibitor added. A decrease from 100% totalenzyme activity is considered a positive response.

A dose dependent response towards reduction of collagenase activity wasobserved for both the pomegranate extract and the grape seed extract(Table 15).

Activated osteoclasts reduce bone strength and increase bone loss bysecreting matrix digesting enzymes (MMP's) to break down the bone'scollagen/calcium phosphate framework. By the mechanism suggested here,the grape seed and pomegranate extracts in particular are found to bepotent inhibitors of collagenase activity. By this activity a netpositive balance of collagen production may be achieved resulting in themaintenance or improvement of bone strength and bone integrity.

TABLE 15 Inhibition of C. histolyticum collagenase activity by GrapeSeed and Pomegranate Extracts % Collagenase Sample Concentration (μg/ml)Activity Pomegranate extract 1  96.7 ± 10.0% 10 86.3 ± 5.9% 100 29.2 ±5.7% Grape seed extract 1 98.9 ± 3.0% 10 48.8 ± 2.3% 100 −7.2 ± 4.3%Collagenase only 0 100.0 ± 0.8% 

Example 9 Calvarial Assay to Measure Inhibition of Bone Resorption

In order to label cells of newborn mice in utero, pregnant CD-1 femalemice (at timed day 15) were injected with ⁴⁵Ca (25 uCi/mouse). Thecalvaria (skull bones) from the 4 day old pups were dissected out andcut in half. The excised half calvaria were placed on metal grids (atthe surface) in 1 ml of BGJ growth medium (Sigma) containing 0.1% BSAwith glutamine and Pen/Strep added. The bones were incubated at 37° C.in a 5% CO₂ humidified incubator for a period of 24 hours followingwhich they were transferred to wells containing 1 ml media with factorsadded (IL-1, PTH and or compounds.) The treated bones were incubatedunder the above conditions for a further 72 hours. After this incubationperiod the bones were removed and placed into 20% TCA in a scintillationvial for 1.5 hours (to measure labeled calcium retained in the bone) andthen counted with scintillation fluid. In addition, 0.4 ml of the mediawas also counted (to determine the amount of labeled calcium releasedfrom the bones). The results were expressed as % ⁴⁵Ca release and can befurther reported as T/C ratios.

For some factors and compounds, modification of this procedure can beused. As most of the osteoclasts are formed in the calvaria followingthe preincubation period, the factors that affect osteoclast formationmay have a greater effect during the preincubation period. Thus, for anumber of the compounds, they were included in the preincubation media.

The extracts tested in this assay include Ginkgo biloba extract, greentea extract, Sophora fructus japonica extract, Rehmannia sp. extract,pomegranate extract, Siberian ginseng, ipriflavone, grape seed extract,and Dong quai extract. The results of testing these extracts in thecalvarial assay are reported below in Table 16.

In Table 16, R-1=Rehmannia sp. (EUL), SFJ=Sophora Fructus Japonica,SG=Siberian Ginseng, SJ=Sophora Japonica (NuPharma), I=Ipriflavone,GB=Ginkgo Biloba, GT=Green Tea, P-1=Pomegranate extract, P-2=Pomegranateextract (Naturex), GS=Grape Seed, DQ=Dong Quai extract. In the belowtable, superscript a (^(a)) means that the result is significantlydifferent (p<0.05) to the blank sample (concentration at 0 μg/ml)result. Differences with p≦0.05 (i.e. where the margin of error is lessthan or equal to 5%) were considered significant. As shown in table 16,pomegranates, ipriflavone, green tea extract, and grape seed extractinhibited IL-1b induced bone resorption/release of calcium.

TABLE 16 Calvarial Data - Inhibition of Bone Resorption/Release of Ca²⁺Conc. R-1 SFJ SG SJ I GB L-1 + μg/ml % Release IL-1 + 100 μg/ml 35.50 ±2.18 30.50 ± 1.85 38.25 ± 1.93 34.50 ± 2.78 19.25 ± 1.03^(a) 33.50 ±2.78 IL-1 + 10 μg/ml 25.70 ± 1.49 29.00 ± 2.74 41.25 ± 1.11 31.70 ± 1.1132.50 ± 3.30 34.50 ± 3.52 IL-1 + 1 μg/ml 31.50 ± 0.96 28.25 ± 2.25 39.25± 1.38 33.00 ± 3.42 38.00 ± 1.41 37.50 ± 1.44 IL-1 28.70 ± 2.56 28.70 ±2.56 45.70 ± 1.65 32.50 ± 3.18 40.00 ± 2.12 31.50 ± 2.10 No IL-1 9.50 ±0.65 9.50 ± 0.65 14.75 ± 2.40 10.50 ± 0.65 10.50 ± 0.85 11.25 ± 1.32Conc. GT P-1 P-2 GS DQ L-1 + μg/ml % Release IL-1 + 100 μg/ml 11.00 ±0.71^(a) 12.00 ± 1.41^(a) 8.75 ± 0.25^(a) 13.75 ± 1.41^(a) 38.50 ± 1.32IL-1 + 10 μg/ml 36.00 ± 2.04 25.50 ± 1.71^(a) 25.25 ± 2.36^(a) 42.75 ±1.71 37.25 ± 2.14 IL-1 + 1 μg/ml 40.25 ± 2.29 39.00 ± 2.97 36.50 ±1.85^(a) 37.75 ± 2.97 40.50 ± 1.55 IL-1 31.50 ± 2.10 38.25 ± 0.25 45.70± 1.65 40.00 ± 0.25 38.25 ± 0.25 No IL-1 11.20 ± 1.32 15.75 ± 1.80 14.70± 2.43 10.25 ± 1.80 15.75 ± 1.70

The calvarial assay described above was repeated with variouscombinations of the extracts useful in compositions of the presentinvention. The results for each formulation are reported below in Table16.

In Table 17, P=Pomegranate, I=Ipriflavone, GS=Grape Seed, GT=Green Tea.Alendronate was used as a positive control. In the below table,superscript a (^(a)) means that the result is significantly different(p<0.05) to the blank sample (concentration at 0 μg/ml) result.Differences with p≦0.05 (i.e. where the margin of error is less than orequal to 5%) were considered significant. As shown in Table 17, alltested combinations inhibited IL-1b induced bone resorption/release ofcalcium.

TABLE 17 Calvarial Data - Inhibition of Bone Resorption/Release ofCalcium By Combinations of Extracts P:GS:I P:GS P:GT P:GS:GT P:I:GT IConc. Alendronate Conc. 500:50:600 1000:100 1000:100 1000:100:100500:600:50 600 μg/ml % Release μg/ml % Release IL-1 + 12.00 ± 0.17^(a)IL-1 + 9.00 ± 0.50^(a) 9.00 ± 0.41^(a) 9.75 ± 0.48^(a) 9.00 ± 0.41^(a)9.50 ± 0.29^(a) 17.50 ± 1.66^(a) 100 μM 100 μg/ml IL-1 + 14.07 ±1.05^(a) IL-1 + 26.00 ± 2.06^(a) 22.25 ± 1.44^(a) 22.25 ± 2.14^(a) 21.25± 1.80^(a) 23.50 ± 2.06^(a) 30.25 ± 4.03 10 μM 10 μg/ml IL-1 + 17.00 ±1.15^(a) IL-1 + 40.75 ± 4.48 34.0 ± 3.70 36.25 ± 4.94 32.75 ± 3.82 44.50± 0.96 29.75 ± 4.54 1 μM 1 μg/ml IL-1 + 28.50 ± 1.19 IL-1 35.50 ± 3.0135.50 ± 3.01 30.25 ± 1.97 30.25 ± 1.97 36.255 ± 2.18 36.25 ± 2.18 0.1 μMIL-1 30.00 ± 0.99 No IL-1 10.50 ± 0.65 10.50 ± 0.65 11.25 ± 0.75 11.25 ±0.75 13.00 ± 1.08 13.00 ± 1.08 No IL-1 10.00 ± 0.78

Example 10 In Vivo Study to Measure Inhibition of RANK-L and Inhibitionof Bone Resorption

Two different formulas were tested in an in vivo study involving female,intact rats, aged approximately 12-14 weeks, with approximate bodyweights between 200-250 grams. Formula 1 included varying dosages ofipriflavone, pomegranate extract, and grape seed extract as reported inTable 18. Formula 2 included varying dosages of pomegranate extract andgrape seed extract and a fixed dosage of ipriflavone as reported inTable 18. Three dosages were tested for each formula. The study utilized8 groups of rats, with 15 rats in each group and lasted approximately 35days.

Table 18 below indicates the formula given to each of the 8 test groups.The dosages in Table 18 are based on the recommended human dosages andwere converted to the appropriate corresponding rat dose using the FDAconversion formula: Human equivalent dose (HED, mg/kg)=rat dose inmg/kg×(rat weight in kg/human weight in kg)^(0.33)

HED=ratNOAEL×(W-rat/W-human)^((1-b)) ,b=0.67.

In Table 18, I=ipriflavone, P=pomegranate extract, and GS=grape seedextract.

TABLE 18 Dosages and Formulas For Analysis In Vivo Group #: Formula:Dosage (mg): 1 P + GS P = 500, GS = 50 2 P + GS P = 1250, GS = 125 3 P +GS P = 2000, GS = 200 4 P + GS + I P = 0, GS = 0, I = 600, 5 P + GS + IP = 500, GS = 50, I = 600, 6 P + GS + I P = 1250, GS = 125, I = 600, 7Placebo 8 Positive Control (Alendronate - 0.5 mg/day)

To introduce rapid bone resorption caused by estrogen withdrawal, theovariectomized (OVX) rat model was used in this study. See, e.g. Alam etal., “Effects of Safflower Seed Oil in Osteoporosis-inducedOvariectomized Rats,” Am. J. Chinese Medicine, 2006. Vol. 34(4):601-612, the contents of which are incorporated herein by reference. Itwas important to evaluate the effect of bone resorption caused by theovariectomy procedure so a Sham control (n=6), a group of female ratswithout undergoing the ovariectomy procedure, was also included in thestudy.

This study employed a randomized placebo controlled dose responsedesign. A total of 120 ovariectomized rats (n=15 per group) aged ˜12-14weeks, with body weights of approximately 200-250 grams were randomlyassigned to groups for 35 days. Animals were fed at 15 grams/day, whichincluded either a placebo diet or one of the formulas listed above, fora period of 35 days from the beginning of the experiment. Chow was madeavailable each morning to the rats at the rate of 15 grams/rat/day. Therats were allowed free access to water and housed in appropriate cagesfor the entire experiment. Unrestricted activity was allowed during theentire experiment.

The effectiveness of the formulas on antiresorption was assessed byoutcome measurements that included DEXA scan (to determine bone mineraldensity), biomechanical measurement (to gather bone strength andfunctional information), micro-CT measurement (to assess virtual bonequality and functional information), and histomorphology measurement (toassess bone quality and functional information). Methods of conducting ahistomorphological measurement are described by Pa Revell,“Histomorphometry of Bone,” J. Clin. Pathol., 1983. Vol. 36: 1321-1331,the entire contents of which are incorporated herein by reference. Theterminology and units used are those recommended by the HistomorphometryNomenclature Committee of the American Society for Bone and MineralResearch. Methods of conducting a micro-CT measurement are described atJiang et al., “Micro CT and Micro MR imagining of 3D architecture ofanimal skeleton.” J. Musculoskel Neuron Interact. 2000. Vol. 1:45-51,the entire contents of which are incorporated herein by reference. Thesemeasurements were determined after 35 days of treatment.

Both DEXA scan and micro-CT measurement results indicated substantialloss of bone mineral density in ovariectomized (OVX) rats compared tosham controlled (non ovariectomized) rats. Bone strength was alsosignificantly reduced as indicated by maximum force to fracture bonesand bone stiffness measurements, as well as % bone volume/total volumeand trabecular cell number in OVX rats. In addition, a marked increasein the trabecular separation (an average of how far the trabecular areaway from each other) was also observed in OVX rats. The positivecontrol group, ALD, was treated with alendronate drug, an effectiveanti-resorption agent known to reduce osteoclastogenesis. See, e.g.,Iwamoto et al., “Comparative effects of alendronate and alfacalcidol oncancellous and cortical bone mass and bone mechanical properties inovariectomized rats.” Exp. Anim. 2006. vol. 55(4):357-67, the entirecontents of which are incorporated herein by reference. The positivecontrol group (ALD) experienced increases in bone mineral density, %bone volume/total volume, and trabecular number and experienced a markedreduction in trabecular separation.

One of the efficacy determination techniques used in this study wasmicro-CT measurement, which provides virtual bone quality, structure andfunctional information. As shown below in Table 19, after 35 days oftreatment, the formulas administered to groups 2, 3, 4, 5, and 6 weremore efficacious than placebo (null treatment) in preventing boneresorption. The outcome of bone mineral density (BMD), bone volume,trabecular cell number and trabecular separation correlates with bonehealth. Superscript a (^(a)) means the result significantly differentfrom the placebo group. Differences of p<0.05 are consideredsignificant.

TABLE 19 Histomorphological Measurements Trabecular Bone Mineral % BoneVolume/ Number Trabecular Group # Density Total Volume (N/6 mm³)Separation 1  21% 107%^(a)  58%^(a) −47%^(a) 2  32%^(a) 132%^(a) 99%^(a) −52%^(a) 3  32%^(a) 145%^(a) 122%^(a) −64%^(a) 4  25%^(a)137%^(a) 117%^(a) −58%^(a) 5  22% 156%  96% −42% 6  1%  11%  14% −18%ALD 124%^(a) 632%^(a) 313%^(a) −88%^(a) Sham  41%^(a) 374%^(a) 281%^(a)−82%^(a)

In Table 20, results from the bone mineral density indicated that theformulas administered to groups 2, 3, and 4 effectively reducedosteoclastogenesis and increased bone mineral density. Results alsoindicated that the formulas administered to groups 1, 2, 3, and 4effectively reduced osteoclastogenesis, increased bone volume increasedthe number of trabecular cells and reduced trabecular cell separation.Superscript a (^(a)) means the result significantly different from theplacebo group. Differences of p<0.05 are considered significant.

TABLE 20 DEXA Measurements Group # Bone Mineral Density 1  0% 2  7%^(a)3 10%^(a) 4  5%^(a) 5  5%^(a) 6  3%^(a) ALD 35%^(a) Sham 14%^(a)

Histomorphology measurement was also used as a tool to determine theefficacy of formulas 1 and 2 where trabecular bone volume from eachgroup was quantified. The results from this analysis are reported inTable 21. Superscript a (^(a)) indicates that the result issignificantly different from the placebo group. Differences with p<0.05are considered significant.

As shown in Table 21, formula 1 caused a significant increase intrabecular bone volume (61.4%) compared to the placebo group. Theincrease in trabecular bone volume while observed, the magnitude wassmaller in groups 2 (13.5%) and 3 (13.1%).

TABLE 21 Histomorphological Measurements Group # % Bone Volume 1 61.4%^(a) 2  13.5% 3  13.1%^(a) 4 −12.9% 5  6.4% 6  41.7% ALD214.1%^(a) Sham 127.9%^(a)

Besides bone mass and bone volume evaluation, bone strength was examinedmechanically by two different parameters. One test was conducted todetermine the maximum force needed to fracture the bone and the otherwas to determine the stiffness (the elasticity) of bone. Methods ofdetermining bone strength and stiffness are described by Sturmer et al.,“Standardized Bending and Breaking Test for the Normal and OsteoporoticMetaphyseal Tibias of the Rat: Effect of Estradiol, Testosterone, andRaloxifene,” J. Bone and Mineral Research, 2006. Vol. 21(1):89-96, theentire contents of which are incorporated herein by reference. Theresults of this analysis are reported below in Table 22. Superscript a(^(a)) means the result significantly different from the placebo group.Differences with p<0.05 are considered significant.

Results listed in table 22 indicate that formula 1 as administered togroups 2 and 3 resulted in an 8.3% and 8.1% increase in maximum forcerequired to fracture bones as compared to the placebo group. Formula 1as administered to group 3 resulted in a 16.0% increase in bonestiffness relative to the placebo treatment.

TABLE 22 Biomechanical Measurements Maximum Force Required Group # toFracture Bones Bone Stiffness 1 −0.4  4.9 2   8.3^(a)  0.6 3   8.1^(a)16.0^(a) 4 −0.6  4.6 5   0.9 10.2 6   3.0  3.3 ALD −0.4  0.2 Sham −0.3 6.0

Results from the DEXA scan, biomechanical measurement, micro-CTmeasurement, and histomorphology measurement clearly illustrated thatFormula 1, comprised of pomegranate and grape seed extract, exhibited anability to improve bone structure and architecture and increase bonestrength during estrogen withdrawal.

It is intended that the foregoing detailed description be regarded asillustrative rather than limiting, and that it be understood that it isthe following claims, including all equivalents, that are intended todefine the spirit and scope of this invention.

1. A composition for increasing bone growth comprising a combination ofquercetin and an extract of licorice, wherein the ratio of the amount ofquercetin to the amount of the extract of licorice is within the rangeof 1:1 to 2:1.
 2. The composition of claim 1, wherein the combinationcomprises 10-1000 mg of quercetin and 10-500 mg of an extract oflicorice.
 3. The composition of claim 2, wherein the combinationcomprises 250 mg of quercetin and 125 mg of an extract of licorice. 4.The composition of claim 1, wherein the extract of licorice is anethanol extract.
 5. The composition of claim 4, wherein the compositionincreases bone morphogenetic protein-2 promoter activity, geneexpression, or protein expression.
 6. The composition of claim 1,further comprising one or more of an extract of Siberian ginseng and anextract of Sophora japonica.
 7. The composition of claim 1, wherein theratio of the amount of quercetin to the amount of the extract oflicorice is 1:1.
 8. The composition of claim 1, wherein the ratio of theamount of quercetin to the amount of the extract of licorice is 2:1. 9.A dietary supplement for increasing bone growth and inhibiting boneresorption comprising a combination of: quercetin, an extract oflicorice; an extract of pomegranate and an extract of grape seed,wherein the ratio of the amount of quercetin to the amount of theextract of licorice is within the range of 1:1 to 2:1.
 10. The dietarysupplement of claim 9, comprising 10-1000 mg of quercetin, 10-500 mg ofan extract of licorice, 10-2000 mg of an extract of pomegranate and35-250 mg of an extract of grape seed.
 11. The dietary supplement ofclaim 9, wherein the ratio of the amount of quercetin to the amount ofthe extract of licorice is 1:1.
 12. The dietary supplement of claim 9,wherein the ratio of the amount of quercetin to the amount of theextract of licorice is 2:1.
 13. The dietary supplement of claim 9,wherein administering the dietary supplement to the subject results inone or more of the following: increased bone mineral density, increasedbone volume, increased trabecular cell number, decreased trabecularseparation, increased bone thickness, increased bone mineral appositionrate, increased bone formation rate, improved bone architecture,increased bone strength and increased bone hardness.